U.S. patent application number 11/678487 was filed with the patent office on 2008-10-30 for compositions and methods useful for culturing differentiable cells.
This patent application is currently assigned to NOVOCELL, INC.. Invention is credited to Allan J. Robins, Thomas C. Schulz.
Application Number | 20080268534 11/678487 |
Document ID | / |
Family ID | 38459762 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080268534 |
Kind Code |
A1 |
Robins; Allan J. ; et
al. |
October 30, 2008 |
COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE
CELLS
Abstract
The present invention relates to cell culture methods and
compositions that are essentially serum-free and comprise a basal
salt nutrient solution and an ErbB3 ligand.
Inventors: |
Robins; Allan J.; (Athens,
GA) ; Schulz; Thomas C.; (Athens, GA) |
Correspondence
Address: |
SUTHERLAND ASBILL & BRENNAN LLP
999 PEACHTREE STREET, N.E.
ATLANTA
GA
30309
US
|
Assignee: |
NOVOCELL, INC.
San Diego
CA
|
Family ID: |
38459762 |
Appl. No.: |
11/678487 |
Filed: |
February 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60776113 |
Feb 23, 2006 |
|
|
|
Current U.S.
Class: |
435/363 ;
435/375; 435/383; 435/384; 435/404; 435/405 |
Current CPC
Class: |
C12N 2501/235 20130101;
G01N 33/5073 20130101; C12N 2501/195 20130101; C12N 5/0606
20130101; G01N 33/5008 20130101; C12N 2501/155 20130101; C12N
2501/105 20130101; C12N 2502/13 20130101; C12N 2501/16 20130101;
C12N 2500/90 20130101; C12N 2501/115 20130101; C12N 2509/00
20130101 |
Class at
Publication: |
435/363 ;
435/404; 435/405; 435/375; 435/383; 435/384 |
International
Class: |
C12N 5/06 20060101
C12N005/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Part of the work performed during development of this
invention utilized U.S. Government funds from National Institutes
of Health Grant No. 5 R24 RR021313-05. The U.S. Government has
certain rights in this invention.
Claims
1. A composition comprising a basal salt nutrient solution and an
ErbB3 ligand, said composition being essentially free of serum.
2. The composition of claim 1, wherein said composition is free of
exogenous insulin and insulin substitutes.
3. The composition of claim 2, further comprising insulin-like
growth factor or a functional fragment thereof.
4. The composition of claim 2, wherein said ErbB3 ligand is
selected from the group consisting of Neuregulin-1,
Heregulin-.beta. (HRG-.beta.), Heregulin-.alpha. (HRG-.alpha.), Neu
differentiation factor (NDF), acetylcholine receptor-inducing
activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived
factor (SMDF), Neuregulin-2, Neuregulin-2.beta. (NRG2-.beta.),
Epiregulin, Biregulin and functional fragments thereof.
5. The composition of claim 4, wherein said ErbB3 ligand is
HRG-.beta. or a functional fragment thereof.
6. The composition of claim 5, further comprising transforming
growth factor beta (TGF-.beta.), a TGF-.beta. family member or a
functional fragment thereof.
7. The composition of claim 6, wherein said TGF-.beta. family
member is selected from the group consisting of Nodal, Activin A,
Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic
protein-4 (BMP4), and functional fragments thereof.
8. The composition of claim 7, comprising Activin A.
9. The composition of claim 8, further comprising insulin-like
growth factor or a functional fragment thereof.
10. The composition of claim 9, wherein said composition is free of
exogenous fibroblast growth factor.
11. The composition of claim 9, further comprising at least one
fibroblast growth factor (FGF) selected from the group consisting
of FGF-2, FGF-7, FGF-10, FGF-22 and variants functional fragments
thereof.
12. The composition of claim 11, wherein said at least one FGF is
FGF-7, FGF-10 and FGF-22.
13. The composition of claim 12, further comprising a serum albumin
(SA).
14. The composition of claim 13, wherein the SA is bovine SA (BSA)
or human SA (HSA).
15. The composition of claim 14, wherein the concentration of the
SA is more than about 0.2%, volume to volume (v/v).
16. The composition of claim 14, wherein the concentration of SA is
less than about 5% v/v.
17. A composition comprising a basal salt nutrient solution and a
means for stimulating ErbB2-directed tyrosine kinase activity in
differentiable cells.
18. The composition of claim 17, wherein said means for stimulating
ErbB2-directed tyrosine kinase activity comprises at least one
ligand that specifically binds ErbB3 in said differentiable
cells.
19. The composition of claim 18, wherein the differentiable cells
are primate embryonic stem cells.
20. A method of culturing differentiable cells, said method
comprising a) plating the differentiable cells on a cell culture
surface; b) providing a basal salt nutrient solution to said
differentiable cells; and c) providing a ligand that specifically
binds ErbB3.
21. The method of claim 20, wherein the differentiable cells are
primate embryonic stem cells.
22. The method of claim 21, wherein neither insulin nor an insulin
substitute is provided to said embryonic stem cells.
23. The method of claim 21, further comprising providing
insulin-like growth factor or a functional fragment thereof to said
embryonic stem cells.
24. The method of claim 23, wherein said ligand that specifically
binds ErbB3 is selected from the group consisting of Neuregulin-1,
Heregulin-.beta. (HRG-.beta.), Heregulin-.alpha. (HRG-.alpha.), Neu
differentiation factor (NDF), acetylcholine receptor-inducing
activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived
factor (SMDF), Neuregulin-2, Neuregulin-2.beta. (NRG2-.beta.),
Epiregulin, Biregulin and functional fragments thereof.
25. The method of claim 24, wherein said ligand is HRG-.beta. or a
functional fragment thereof.
26. The method of claim 25, further comprising providing to said
cells transforming growth factor beta (TGF-.beta.), a TGF-.beta.
family member or a functional fragment thereof.
27. The method of claim 26, wherein said TGF-.beta. family member
is selected from the group consisting of Nodal, Activin A, Activin
B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4
(BMP4), and functional fragments thereof.
28. The method of claim 27, further comprising providing
insulin-like growth factor or a functional fragment thereof to said
embryonic stem cells.
29. The method of claim 28, further comprising providing at least
one fibroblast growth factor (FGF) selected from the group
consisting of FGF-2, FGF-7, FGF-10, FGF-22 and functional fragments
thereof to said embryonic stem cells.
30. The method of claim 29, wherein said at least one FGF is FGF-7,
FGF-10 and FGF-22.
31. The method of claim 30, further comprising providing a serum
albumin (SA) to said embryonic stem cells.
32. The method of claim 31, wherein the SA is bovine SA (BSA) or
human SA (HSA).
33. The method of claim 32, wherein the concentration of the SA is
more than about 0.2%, volume to volume (v/v).
34. The method of claim 33, wherein the concentration of SA is less
than about 5% v/v.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
60/776,113, filed 23 Feb. 2006, which is incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to cell culture methods and
compositions that are essentially serum-free and comprise a basal
salt nutrient solution and an ErbB3 ligand.
[0005] 2. Background of the Invention
[0006] Human pluripotent cells offer unique opportunities for
investigating early stages of human development as well as for
therapeutic intervention in several disease states, such as
diabetes mellitus and Parkinson's disease. For example, the use of
insulin-producing .beta.-cells derived from human embryonic stem
cells (hESCs) would offer a vast improvement over current cell
therapy procedures that utilize cells from donor pancreases.
Currently cell therapy treatments for diabetes mellitus, which
utilize cells from donor pancreases, are limited by the scarcity of
high quality islet cells needed for transplant. Cell therapy for a
single Type I diabetic patient requires a transplant of
approximately 8.times.10.sup.8 pancreatic islet cells (Shapiro et
al., 2000, N Engl J Med 343:230-238; Shapiro et al, 2001a, Best
Pract Res Clin Endocrinol Metab 15:241 264; Shapiro et al, 2001,
British Medical Journal 322:861). As such, at least two healthy
donor organs are required to obtain sufficient islet cells for a
successful transplant.
[0007] Embryonic stem (ES) cells thus represent a powerful model
system for the investigation of mechanisms underlying pluripotent
cell biology and differentiation within the early embryo, as well
as providing opportunities for genetic manipulation of mammals and
resultant commercial, medical and agricultural applications.
Furthermore, appropriate proliferation and differentiation of ES
cells can potentially be used to generate an unlimited source of
cells suited to transplantation for treatment of diseases that
result from cell damage or dysfunction. Other pluripotent cells and
cell lines including early primitive ectoderm-like (EPL) cells as
described in International Patent Application WO 99/53021, in vivo
or in vitro derived ICM/epiblast, in vivo or in vitro derived
primitive ectoderm, primordial germ cells (EG cells),
teratocarcinoma cells (EC cells), and pluripotent cells derived by
dedifferentiation or by nuclear transfer will share some or all of
these properties and applications. International Patent Application
WO 97/32033 and U.S. Pat. No. 5,453,357 describe pluripotent cells
including cells from species other than rodents. Human ES cells
have been described in International Patent Application WO
00/27995, and in U.S. Pat. No. 6,200,806, and human EG cells have
been described in International Patent Application WO 98/43679.
[0008] The biochemical mechanisms regulating ES cell pluripotency
and differentiation are very poorly understood. However, the
limited empirical data available (and much anecdotal evidence)
suggests that the continued maintenance of pluripotent ES cells
under in vitro culture conditions is dependent upon the presence of
cytokines and growth factors present in the extracellular
milieu.
[0009] While human ESCs offer a source of starting material from
which to develop substantial quantities of high quality
differentiated cells for human cell therapies, these cells must be
obtained and/or cultured in conditions that are compatible with the
expected regulatory guidelines governing clinical safety and
efficacy. Such guidelines likely will require the use of a
chemically defined media. The development of such chemically
defined/GMP standard conditions is necessary to facilitate the use
of hESCs and cells derived from hESCs for therapeutic purposes in
humans.
[0010] In addition, the eventual application of hESC based cell
replacement therapies will require the development of methods that
enable large scale culture and differentiation conditions that are
compliant with regulatory guidelines. While several groups have
reported simplified growth conditions for hESCs, there are
substantial limitations with these studies. To date, however, the
successful isolation, long-term clonal maintenance, genetic
manipulation and germ line transmission of pluripotent cells has
generally been difficult.
[0011] Most of the cell culture conditions for stem cells still
contain serum replacer (KSR) in the media (Xu et al., 2005 Stem
Cells, 23:315-323; Xu et al., 2005 Nature Methods, 2:185-189;
Beattie et al., 2005 Stem Cells, 23:489-495; Amit et al., 2004
Biol. Reprod., 70:837-845; James et al., 2005 Development,
132:1279-1282). KSR contains a crude fraction of bovine serum
albumin (BSA) rather than a highly purified source. Others have
only performed short-term studies, and therefore it is not clear if
their conditions would enable the maintenance of pluripotency over
extended periods (Sato et al., (2004) Nature Med., 10:55-63; U.S.
Patent Publication Nos. 2006/0030042 and 2005/0233446). Others have
shown long-term maintenance of pluripotency in a chemically defined
media with FGF2, activin A, and insulin, but the cells were grown
on plates that were coated with human serum, which was "washed off"
before plating of cells (Vallier et al., 2005 J Cell Sci., 118(Pt
19):4495-509). While FGF2 has been a component of all these media,
it is not clear if it is an absolute necessity, particularly as in
some formulations it is necessary to use it at a high concentration
(up to 100 ng/ml, Xu et al., 2005 Nature Methods, 2:185-189).
[0012] Furthermore, all of these groups have either included
insulin in their media at .mu.g/ml levels, or have insulin present
due to the use of KSR. Insulin is typically considered to function
in glucose metabolism and "cell survival" signaling via binding to
the insulin receptor. At levels above physiological concentrations,
however, insulin can also bind to the IGF1 receptor with a lower
efficiency and confer classical growth factor activity through the
PI3 Kinase/AKT pathway. The presence/requirement for such high
levels of insulin (.mu.g/ml levels) in KSR or these other media
conditions suggests that the major activity is elicited via binding
to the IGF1 receptor, which is expressed by hESCs (Sperger et al.,
2003 PNAS, 100(23):13350-13355). Others have noted the expression
of a full complement of IGF1R and intracellular signaling pathway
members in hESCs, which is likely to signify the functional
activity of this pathway (Miura et al., 2004 Aging Cell,
3:333-343). Insulin or IGF1 may elicit a major signal required for
the self-renewal of hESCs, as is suggested by the fact that all
conditions developed thus far for the culture of hESC contain
either insulin, insulin provided by KSR, or IGF1 provided by serum.
In support of this concept, it has been shown that if PI3 Kinase is
inhibited in hESC cultures, the cells differentiate (D'Amour et
al., 2005 Nat. Biotechnol., 23(12):1534-41; McLean et al., 2007
Stem Cells 25:29-38).
[0013] A recent publication outlines a humanized, defined media for
hESCs (Ludwig et al., Nature Biotechnology, published online Jan.
1, 2006, doi:10.1038/nbt1177). This recent formulation, however,
includes several factors that are suggested to influence the
proliferation of hESCs, including FGF2, TGF.beta., LiCl,
.gamma.-aminobutyric acid and pipecolic acid. It is noted that this
recently defined cell culture medium also contains insulin.
[0014] The EGF growth factor family has at least 14 members,
including, but not limited to, EGF, TGF.beta., heparin binding-EGF
(hb-EGF), neuregulin-.beta. (also named heregulin-.beta.
(HRG-.beta.), glial growth factor and others), HRG-.alpha.,
amphiregulin, betacellulin, and epiregulin. All these growth
factors contain an EGF domain and are typically first expressed as
transmembrane proteins that are processed by metalloproteinase
(specifically, ADAM) proteins to generate soluble ectodomain growth
factors. EGF family members interact with either homo- or
hetero-dimers of the ErbB1, 2, 3 and 4 cell surface receptors with
different affinities (Jones et al., FEBS Lett, 1999, 447:227-231).
EGF, TGF.alpha. and hbEGF bind ErbB1/1 (EGFR) homodimers and
ErbB1/2 heterodimers at high affinity (1-100 nM range), whereas
HRG-.beta. binds ErbB3 and ErbB4 at very high affinity (<1 nM
range). Activated ErbB receptors signal through the PI3 Kinase/AKT
pathway and also the MAPK pathway. ErbB2 and ErbB3 are amongst the
most highly expressed growth factor receptors in hESCs (Sperger et
al., 2003 PNAS, 100(23):13350-13355) and HRG-.beta. has been shown
previously to support the expansion of mouse primordial germ cells
(Toyoda-Ohno et al., 1999 Dev. Biol., 215(2):399-406). Furthermore,
overexpression and subsequent inappropriate activation of ErbB2 is
associated with tumorigenesis (Neve et al., 2001 Ann. Oncol., 12
Suppl 1:S9-13; Zhou & Hung, 2003 Semin. Oncol., 30(5 Suppl
16):38-48; Yarden, 2001 Oncology, 61 Suppl 2:1-13). Human ErbB2
(Chromosome 17q), and ErbB3 (Chromosome 12q) are present on
chromosomes that have been observed to accumulate as trisomies in
some hESCs (Draper et al., 2004 Nat. Biotechnol., 22(1):53-4; Cowan
et al., 2004 N Engl. J. Med., 350(13):1353-6; Brimble et al., 2004
Stem Cells Dev., 13(6):585-97; Maitra et al., 2005 Nat. Genet.
37(10):1099-103; Mitalipova et al., 2005 Nat. Biotechnol. 23(1):
19-20; Draper et al., 2004 Stem Cells Dev., 13(4):325-36; Ludwig et
al., Nature Biotechnology, published online Jan. 1, 2006,
doi:10.1038/nbt1177).
[0015] ErbB2 and ErbB3 (Brown et al., 2004 Biol. Reprod.,
71:2003-2011; Salas-Vidal & Lomeli, 2004, Dev Biol., 265:75-89)
are expressed in the mouse blastocyst, although not specifically
restricted to the inner cell mass (ICM), and ErbB1, EGF and
TGF.beta. are expressed in the human blastocyst (Chia et al., 1995
Development, 1221(2):299-307). HB-EGF has proliferative effects in
human IVF blastocyst culture (Martin et al., 1998 Hum. Reprod.,
13(6): 1645-52; Sargent et al., 1998 Hum. Reprod. 13 Suppl
4:239-48), and modest additional effects on mouse ES cells grown in
15% serum (Heo et al., 2005, Am. J. Phy. Cell Physiol., in press).
Pre- and early post-implantation development does not appear to be
affected in ErbB2-/-, ErbB3-/-, Neuregulin1-/- (Britsch et al.,
1998 Genes Dev., 12:1825-36), ADAM17-/- (Peschon, et al., 1998
Science, 282: 1281-1284) and ADAM19-/- (Horiuchi, 2005 Dev. Biol.,
283(2):459-71) null embryos. Therefore, the importance of signaling
through the ErbB receptor family in hESCs is, up to now,
unclear.
[0016] Neuregulin-1 (NRG1) is a large gene that exhibits multiple
splicing and protein processing variants. This generates a large
number of protein isoforms, which are referred to herein
collectively as neuregulin. Neuregulin is predominantly expressed
as a cell surface transmembrane protein. The extracellular region
contains an immunoglobulin-like domain, a carbohydrate modified
region and the EGF domain. NRG1 expression isoforms have been
reviewed previously (Falls, 2003 Exp. Cell Res., 284: 14-30). The
cell membrane metalloproteases ADAM17 and ADAM19 have been shown to
process the transmembrane form(s) of neuregulin-1 to soluble
neuregulin/heregulin. HRG-.alpha. and -.beta. are the cleaved
ectodomains of neuregulin, containing the EGF and other domains. As
the EGF domain is responsible for binding and activation of the
ErbB receptors, a recombinant molecule containing only this domain
can exhibit essentially all of the soluble growth factor effects of
this protein (Jones et al., 1999 FEBS Lett., 447:227-231). Also,
there are processed transmembrane isoforms of neuregulin that are
thought to trigger juxtacrine signaling in adjacent cells via
interaction of the EGF domain with ErbB receptors.
[0017] An important development in the progression of hESC research
toward maintaining pluripotency in culture will be the elucidation
of media and cell culture conditions that are compatible with the
expected regulatory guidelines governing clinical safety and
efficacy. While the best outcome would be the availability of
chemically defined media for hESCs, components that are not
chemically defined would be acceptable if they were produced to GMP
standard. There is a need, therefore, to identify methods and
compositions for the culture and stabilization of a population of
pluripotent stem cells that are able to be used for therapeutic
purposes, wherein the culture compositions are defined and/or
produced to GMP standard.
SUMMARY OF THE INVENTION
[0018] The invention relates to compositions comprising a basal
salt nutrient solution and an ErbB3 ligand, with the compositions
being essentially free of serum.
[0019] The invention also relates to compositions comprising a
basal salt nutrient solution and a means for stimulating
ErbB2-directed tyrosine kinase activity in differentiable
cells.
[0020] The invention relates to methods of culturing differentiable
cells, with the methods comprising plating the differentiable cells
on a cell culture surface, providing a basal salt nutrient solution
to the differentiable cells and providing a ligand that
specifically binds ErbB3.
[0021] The invention relates to methods of culturing differentiable
cells, with the methods comprising plating the differentiable cells
on a cell culture surface and providing a basal salt nutrient
solution to the differentiable cells and a means for stimulating
ErbB2-directed tyrosine kinase activity in the differentiable
cells.
[0022] The invention also relates to methods of culturing
differentiable cells, with the methods comprising providing a
digest solution to a layer of differentiable cells that are
contained in a culture chamber prior to digestion, where the
digestion breaks apart the layer of cells into single cells. After
digestion, the single cells are placed into a new tissue culture
chamber with a differentiable cell culture solution, wherein the
differentiable cell culture solution comprises a basal salt
nutrient solution and an ErbB3 ligand. Once cultured, the single
differentiable cells are placed in conditions that permit growth
and division of the single cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts real time RT-PCR expression analysis of
ADAM19, Neuregulin1, and ErbB1-3 in BG01v grown in defined
conditions (8 ng/ml FGF2, 100 ng/ml LR-IGF1, 1 ng/ml Activin A).
GAPDH and OCT4 control reactions are indicated.
[0024] FIG. 2 depicts the inhibition of proliferation of BG01v
cells using AG879. BG01v cells were plated in 6-well trays and
exposed to DMSO (A), 50 nM-20 .mu.M AG1478 (B), or 100 mM-20 .mu.M
AG879 (C) 24 hours after plating. After 5 days in culture, the
cultures were fixed and stained for alkaline phosphatase activity.
AG1478 did not appear to affect proliferation at these
concentrations (20 .mu.M shown in B), but AG879 substantially
slowed cell growth at 5 .mu.M (C).
[0025] FIG. 3 depicts the morphology of BG01v cells cultured in
DC-HAIF, which is defined culture media containing 10 ng/ml
HRG-.beta., 10 ng/ml Activin A, 200 ng/ml LR-IGF1 and 8 ng/ml FGF2
(A and B), and in defined culture media (DC) containing 10 ng/ml
HRG-.beta., 10 ng/ml Activin A, and 200 ng/ml LR-IGF1 (C and
D).
[0026] FIG. 4 depicts the expression of ADAM19, Neuregulin1, and
ErbB1-4 by RT-PCR in mouse ES cells (A) and MEFs (B).
[0027] FIG. 5 depicts the inhibition of ErbB1 and ErbB2 signaling
in mouse ES cells. 2.times.10.sup.5 Mouse R1ES cells were plated on
1:1000 MATRIGEL.TM. in 10% FBS, 10% KSR with 1000 U/ml mouse LIF
(ESGRO). The following day, DMSO (carrier control), 1-50 .mu.M AG
1478, or 1-50 .mu.M AG879 was added with fresh medium. The cultures
were fixed on day 8, and stained for alkaline phosphatase activity.
DMSO (A) and 1-50 .mu.M AG1478 (B and C) did not overtly inhibit
proliferation. AG879 substantially inhibited cell growth at 50
.mu.M (compare D and F) and may have slowed proliferation at 20
.mu.M (E).
[0028] FIG. 6 depicts the inhibition of proliferation of BG02 cells
grown in conditioned media (CM). (A) 50 .mu.M AG825 inhibited
proliferation of BG02 hESCs growing in CM. (B) AG825 inhibits ErbB2
Y1248 phosphorylation in hESCs. (C) Colony counting of serial
passaging of CyT49 hESCs in different combinations of growth
factors. (D) Cell counting analysis of the role of IGF1 and HRG in
hESC proliferation using BG02 cells (left). (E) OCT4/DAPI
immunostaining of a duplicate repeated experiment demonstrated that
IGF1 and HRG significantly increased the proportion of OCT4.sup.+
cells compared to ActA/FGF2 conditions. (F) RTK blotting analysis
of BG01 DC-HAIF hESCs starved of growth factors overnight; starved,
then pulsed with DC-HAIF for 15 minutes; or steady-state cultures
are shown (left). The mean and range of normalized relative
intensity is plotted (right).
[0029] FIG. 7 depicts mouse ES cells grown in defined conditions
with different growth factor combinations. (A) shows the scoring of
AP.sup.+ colonies after 2.times.10.sup.5 cells were grown in
different growth factor combinations for 8 days. (B-G) show
4.times. magnification images of AP.sup.+ colonies grown in
different growth factor combinations.
[0030] FIG. 8 depicts the characterization of human ES cells that
are maintained in DC-HAIF medium. (A) Analysis of teratomas from
BG02 DC-HAIF p25 cells demonstrated pluripotent differentiation
potential to ectoderm, mesoderm and endoderm. (B) Immunostaining of
BG02 cells cultured in 15% FCS/5% KSR that have differentiated. (C)
Venn diagram of the distribution of transcripts detected using high
density Illumina Sentrix Human-6 Expression Beadchips containing
47,296 transcript probes in BG02 cells maintained in CM (64
passages) or DC-HAIF (10 or 32 passages in defined media). (D)
Scatterplot analysis demonstrating that the transcriptional profile
of BG02 DC-HAIF p32 cells is highly similar to that of BG02 cells
maintained in CM (top), and was not substantially altered in early
and late passage cultures in DC-HAIF (bottom). (E) Hierarchical
clustering dendrogram of relative gene expression in different
populations generated using the Beadstudio software.
[0031] FIG. 9 depicts the morphology of cells cultured on humanized
extracellular matrices (ECMs) in the presence of DC-HAIF medium.
(A) CyT49 cells (diluted 1:200) growing on growth factor-reduced
MATRIGEL.TM. (diluted 1:200). CyT49 cells could also grow on tissue
culture dishes coated with (B) whole human serum, (C) human
fibronectin, and (D) VITROGRO.TM..
[0032] FIG. 10 depicts the single-cell passaging of human ES cells.
(A-D) Staged imaging of BG02 cells after passaging with
ACCUTASE.TM. and plating about 5.times.10.sup.5 cells in a 60 mm
culture dish. (A) 1.5 hours after initial plating, showing viable
cells adhering to the dish. (B) At 20 hours post-plating, the large
majority of cells have aggregated to form small colonies. These
colonies expand by proliferation by day 4, post-plating (C), and
over the course of 5-6 days to form an epithelial-like monolayer
covering the entire dish (D). (E) Normal male karyotype
demonstrated in a BG02 culture passaged 19 times with ACCUTASE.TM.
in DC-HAIF.
[0033] FIG. 11 depicts cell morphology after single cell passaging
of human ES cells using (A) ACCUTASE.TM., (B) 0.25% Trypsin/EDTA,
(C) TrypLE, or (D) Versene.
[0034] FIG. 12 depicts the large-scale growth of human ES cells
cultured in DC-HAIF. (A) Flow cytometric analysis of BG02 cells
after expansion to >10.sup.10 cells. >85% of cells expressed
OCT4, CD9, SSEA-4, TRA-1-81. (B) RT-PCR analysis of expression of
markers of pluripotency OCT4, NANOG, REX1, SOX2, UTF1, CRIPTO,
FOXD3, TERT and DPPA5. Markers of differentiated lineages,
.alpha.-fetoprotein (AFP), MSX1 and HAND1 were not detected. (C)
Fluorescence in situ hybridization (FISH) using human
chromosome-specific repeats demonstrated maintenance of normal copy
numbers for hChr 12, 17, X and Y.
[0035] FIG. 13 depicts the morphology (A) and normal karyotype (B)
of hESC BG02 cells grown in defined media comprising HRG-.beta. and
IGF1, but in the absence of FGF2 for 7 passages, or >2
months.
[0036] FIG. 14 depicts a scatterplot analysis of transcripts from
hESCs (BG02) that are maintained in DC-HAIF (32 passages) or DC-HAI
(10 passages). A large proportion of the expressed transcripts were
detected in both samples, and transcription was not substantially
altered by culturing hESCs in the absence of exogenous FGF2.
Correlation coefficients (R.sup.2) were generated using all
detected transcripts with an expression level of >0 (all dots),
or with transcripts exhibiting a detection confidence level of
>0.99 (R.sup.2 select, dots indicated by dashed oval). Angled
lines delineate the mean and limits of a 2-fold difference.
[0037] FIG. 15 depicts a hierarchical clustering dendrogram of
relative gene expression in different populations of early and late
passage BG02 cells maintained in DC-HAIF. Cells clustered tightly
(.about.0.0075) and retained a close similarity to BG02 and BG03
cells maintained in conditioned medium (CM) (.about.0.037). BG02
cells maintained in DC-HAI also clustered tightly with the other
hESC populations examined. By way of explanation in FIG. 15, CM is
Conditioned Medium; DC is defined culture medium, DC-HAIF as
defined above; ap is ACCUTASE.TM. single cell passaging; DC-HAI is
identical to DC-HAIF as defined herein, except without FGF2.
[0038] FIG. 16 depicts the morphology and alkaline phsophatase
staining of BG02 cells cultured in DC-HAIF in 96-well and 384-well
plates. (A) Phase contrast imaging and (B) alkaline phosphatase
staining of BG02 cells (10.sup.4 cells/well) growing in one well of
a 96-well plate. (C) Phase contrast imaging and (D) alkaline
phosphatase staining of BG02 cells (10.sup.3 cells/well) growing in
one well of a 384-well plate.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Unless otherwise noted, the terms used herein are to be
understood according to conventional usage by those of ordinary
skill in the relevant art. In addition to the definitions of terms
provided below, definitions of common terms in molecular biology
may also be found in Rieger et al., 1991 Glossary of genetics:
classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in
Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1998
Supplement). It is to be understood that as used in the
specification and in the claims, "a" or "an" can mean one or more,
depending upon the context in which it is used. Thus, for example,
reference to "a cell" can mean that at least one cell can be
utilized.
[0040] As used herein, the term "contacting" (i.e., contacting a
cell e.g., a differentiable cell, with a compound) is intended to
include incubating the compound and the cell together in vitro
(e.g., adding the compound to cells in culture). The term
"contacting" is not intended to include the in vivo exposure of
cells to a defined cell medium comprising an ErbB3 ligand, and
optionally, a member of the TGF-.beta. family, that may occur
naturally in a subject (i.e., exposure that may occur as a result
of a natural physiological process). The step of contacting the
cell with a defined cell medium comprising an ErbB3 ligand, and
optionally, a member of the TGF-.beta. family, can be conducted in
any suitable manner. For example, the cells may be treated in
adherent culture, or in suspension culture. It is understood that
the cells contacted with the defined medium can be further treated
with a cell differentiation environment to stabilize the cells, or
to differentiate the cells.
[0041] As used herein, the term "differentiate" refers to the
production of a cell type that is more differentiated than the cell
type from which it is derived. The term therefore encompasses cell
types that are partially and terminally differentiated.
[0042] In certain embodiments of the present invention, the term
"enriched" refers to a cell culture that contains more than
approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%
of the desired cell lineage.
[0043] As used herein, the term "effective amount" of a compound
refers to that concentration of the compound that is sufficient in
the presence of the remaining components of the defined medium to
effect the stabilization of the differentiable cell in culture for
greater than one month in the absence of a feeder cell and in the
absence of serum or serum replacement. This concentration is
readily determined by one of ordinary skill in the art.
[0044] As used herein, the term "express" refers to the
transcription of a polynucleotide or translation of a polypeptide
in a cell, such that levels of the molecule are measurably higher
in a cell that expresses the molecule than they are in a cell that
does not express the molecule. Methods to measure the expression of
a molecule are well known to those of ordinary skill in the art,
and include without limitation, Northern blotting, RT-PCR, in situ
hybridization, Western blotting, and immunostaining.
[0045] As used herein when referring to a cell, cell line, cell
culture or population of cells, the term "isolated" refers to being
substantially separated from the natural source of the cells such
that the cell, cell line, cell culture, or population of cells are
capable of being cultured in vitro. In addition, the term
"isolating" is used to refer to the physical selection of one or
more cells out of a group of two or more cells, wherein the cells
are selected based on cell morphology and/or the expression of
various markers.
[0046] The present invention may be understood more readily by
reference to the following detailed description of the preferred
embodiments of the invention and the Examples included herein.
However, before the present compositions and methods are disclosed
and described, it is to be understood that this invention is not
limited to specific nucleic acids, specific polypeptides, specific
cell types, specific host cells, specific conditions, or specific
methods, etc., as such may, of course, vary, and the numerous
modifications and variations therein will be apparent to those
skilled in the art.
[0047] Standard techniques for cloning, DNA isolation,
amplification and purification, for enzymatic reactions involving
DNA ligase, DNA polymerase, restriction endonucleases and the like,
and various separation techniques are those known and commonly
employed by those skilled in the art. A number of standard
techniques are described in Sambrook et al., 1989 Molecular
Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview,
N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor
Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part
I; Wu (Ed.) 1979 Meth. Enzymol. 68; Wu et al., (Eds.) 1983 Meth.
Enzymol. 100 and 101; Grossman and Moldave (Eds.) 1980 Meth.
Enzymol. 65; Miller (ed.) 1972 Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and
Primrose, 1981 Principles of Gene Manipulation, University of
California Press, Berkeley; Schleif and Wensink, 1982 Practical
Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I
and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985
Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and
Hollaender 1979 Genetic Engineering: Principles and Methods, Vols.
1-4, Plenum Press, New York. Abbreviations and nomenclature, where
employed, are deemed standard in the field and commonly used in
professional journals such as those cited herein.
[0048] The invention relates to compositions and methods comprising
a basal salt nutrient solution and an effective amount of an ErbB3
ligand, with the compositions being essentially free of serum. The
compositions and methods of the present invention are useful for
culturing cells, in particular, differentiable cells. It is
understood that at different points during culturing the
differentiable cells, various components may be added to the cell
culture such that the medium can contain components other than
those described herein. It is, however, contemplated that at least
at one point during the preparation of the culture, or during the
culture of the differentiable cells, the defined medium comprises a
basal salt nutrient solution and a means for activating
ErbB2-directed tyrosine kinase.
[0049] As used herein, the term "differentiable cell" is used to
describe a cell or population of cells that can differentiate into
at least partially mature cells, or that can participate in the
differentiation of cells, e.g., fuse with other cells, that can
differentiate into at least partially mature cells. As used herein,
"partially mature cells" are cells that exhibit at least one
characteristic of the phenotype, such as morphology or protein
expression, of a mature cell from the same organ or tissue. For
example, a normal, mature hepatocyte typically expresses such
proteins as albumin, fibrinogen, alpha-1-antitrypsin, prothrombin
clotting factors, transferrin, and detoxification enzymes such as
the cytochrome P-450s, among others. Thus, as defined in the
present invention, a "partially mature hepatocyte" may express
albumin or another one or more proteins, or begin to take the
appearance or function of a normal, mature hepatocyte.
Additionally, a "partially mature pancreatic beta cell" may produce
or express the proinsulin protein, among others. The ability of the
cells to differentiate into at least partially mature cells will
not be dependent upon recombinant engineering techniques, such as
transfection, though the cells may, of course, be genetically
engineered.
[0050] The invention contemplates compositions and methods useful
for differentiable cells, regardless of their source or of their
plasticity. The "plasticity" of a cell is used herein roughly as it
is in the art. Namely, the plasticity of a cell refers to a cell's
ability to differentiate into a particular cell type found in
tissues or organs from an embryo, fetus or developed organism. The
"more plastic" a cell, the more tissues into which the cell may be
able to differentiate. "Pluripotent cells" include cells and their
progeny, which may be able to differentiate into, or give rise to,
pluripotent, multipotent, oligopotent and unipotent cells, and/or
several, if not all, of the mature or partially mature cell types
found in an embryo, fetus or developed organism. "Multipotent
cells" include cells and their progeny, which may be able to
differentiate into, or give rise to, multipotent, oligopotent and
unipotent progenitor cells, and/or one or more mature or partially
mature cell types, except that the mature or partially mature cell
types derived from multipotent cells are limited to cells of a
particular tissue, organ or organ system. For example, a
multipotent hematopoietic progenitor cell and/or its progeny
possess the ability to differentiate into or give rise to one or
more types of oligopotent cells, such as myeloid progenitor cells
and lymphoid progenitor cells, and also give rise to other mature
cellular components normally found in the blood. "Oligopotent
cells" include cells and their progeny whose ability to
differentiate into mature or partially mature cells is more
restricted than multipotent cells. Oligopotent cells may, however,
still possess the ability to differentiate into oligopotent and
unipotent cells, and/or one or more mature or partially mature cell
types of a given tissue, organ or organ system. One example of an
oligopotent cell is a myeloid progenitor cell, which can ultimately
give rise to mature or partially mature erythrocytes, platelets,
basophils, eosinophils, neutrophils and monocytes. "Unipotent
cells" include cells and their progeny that possess the ability to
differentiate or give rise to other unipotent cells and/or one type
of mature or partially mature cell type.
[0051] Differentiable cells, as used herein, may be pluripotent,
multipotent, oligopotent or even unipotent. In certain embodiments
of the present invention, the differentiable cells are pluripotent
differentiable cells. In more specific embodiments, the pluripotent
differentiable cells are selected from the group consisting of
embryonic stem cells, ICM/epiblast cells, primitive ectoderm cells,
primordial germ cells, and teratocarcinoma cells. In one particular
embodiment, the differentiable cells are mammalian embryonic stem
cells. In a more particular embodiment, the differentiable cells
are human embryonic stem cells.
[0052] The invention also contemplates differentiable cells from
any source within an animal, provided the cells are differentiable
as defined herein. For example, differentiable cells may be
harvested from embryos, or any primordial germ layer therein, from
placental or chorion tissue, or from more mature tissue such as
adult stem cells including, but not limited to adipose, bone
marrow, nervous tissue, mammary tissue, liver tissue, pancreas,
epithelial, respiratory, gonadal and muscle tissue. In specific
embodiments, the differentiable cells are embryonic stem cells. In
other specific embodiments, the differentiable cells are adult stem
cells. In still other specific embodiments, the stem cells are
placental- or chorionic-derived stem cells.
[0053] Of course, the invention contemplates using differentiable
cells from any animal capable of generating differentiable cells.
The animals from which the differentiable cells are harvested may
be vertebrate or invertebrate, mammalian or non-mammalian, human or
non-human. Examples of animal sources include, but are not limited
to, primates, rodents, canines, felines, equines, bovines and
porcines.
[0054] The differentiable cells of the present invention can be
derived using any method known to those of skill in the art. For
example, human pluripotent cells can be produced using
de-differentiation and nuclear transfer methods. Additionally, the
human ICM/epiblast cell or the primitive ectoderm cell used in the
present invention can be derived in vivo or in vitro. Primitive
ectodermal cells may be generated in adherent culture or as cell
aggregates in suspension culture, as described in WO 99/53021.
Furthermore, the human pluripotent cells can be passaged using any
method known to those of skill in the art, including, manual
passaging methods, and bulk passaging methods such as enzymatic or
non-enzymatic passaging.
[0055] In certain embodiment, when ES cells are utilized, the
embryonic stem cells have a normal karyotype, while in other
embodiments, the embryonic stem cells have an abnormal karyotype.
In one embodiment, a majority of the embryonic stem cells have a
normal karyotype. It is contemplated that greater than 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 95% of metaphases
examined will display a normal karyotype.
[0056] In another embodiment, a majority of the embryonic stem
cells have an abnormal karyotype. It is contemplated that greater
than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than
95% of metaphases examined will display an abnormal karyotype. In
certain embodiments, the abnormal karyotype is evident after the
cells have been cultured for greater than 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 20 passages. In one specific embodiment, the
abnormal karyotype comprises a trisomy of at least one autosomal
chromosome, wherein the autosomal chromosome is selected from the
group consisting of chromosomes 1, 7, 8, 12, 14, and 17. In another
embodiment, the abnormal karyotype comprises a trisomy of more than
one autosomal chromosome, wherein at least one of the more than one
autosomal chromosomes is selected from the group consisting of
chromosomes 1, 7, 8, 12, 14, and 17. In one embodiment, the
autosomal chromosome is chromosome 12 or 17. In another embodiment,
the abnormal karyotype comprises an additional sex chromosome. In
one embodiment, the karyotype comprises two X chromosomes and one Y
chromosome. It is also contemplated that translocations of
chromosomes may occur, and such translocations are encompassed
within the term "abnormal karyotype." Combinations of the foregoing
chromosomal abnormalities and other chromosomal abnormalities are
also encompassed by the invention.
[0057] The compositions and methods comprise a basal salt nutrient
solution. As used herein, basal salt nutrient solution refers to a
mixture of salts that provide cells with water and certain bulk
inorganic ions essential for normal cell metabolism, maintain
intra- and extra-cellular osmotic balance, provide a carbohydrate
as an energy source, and provide a buffering system to maintain the
medium within the physiological pH range. Examples of basal salt
nutrient solutions include, but are not limited to, Dulbecco's
Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM),
Basal Medium Eagle (BME), RPM1 1640, Ham's F-10, Ham's F-12,
.alpha.-Minimal Essential Medium (.alpha.MEM), Glasgow's Minimal
Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium,
and mixtures thereof. In one particular embodiment, the basal salt
nutrient solution is an approximately 50:50 mixture of DMEM and
Ham's F12.
[0058] It is contemplated that the composition can further comprise
trace elements. Trace elements can be purchased commercially, for
example, from Mediatech. Non-limiting examples of trace elements
include but are not limited to compounds comprising, aluminum,
chlorine, sulfate, iron, cadmium, cobalt, chromium, germanium,
sodium, potassium, calcium, phosphate and magnesium. Specific
example of compounds containing trace elements include but are not
limited to, AlCl.sub.3, AgNO.sub.3,
Ba(C.sub.2H.sub.3O.sub.2).sub.2, CdCl.sub.2, CdSO.sub.4,
CoCl.sub.2, CrCl.sub.3, Cr.sub.2(SO.sub.4).sub.3, CuSO.sub.4,
ferric citrate, GeO.sub.2, KI, KBr, LI, molybdic acid, MnSO.sub.4,
MnCl.sub.2, NaF, Na.sub.2SiO.sub.3, NaVO.sub.3, NH.sub.4VO.sub.3,
(NH.sub.4).sub.6Mo.sub.7O.sub.24, NiSO.sub.4, RbCl, selenium,
Na.sub.2SeO.sub.3, H.sub.2SeO.sub.3, selenite-2Na,
selenomethionone, SnCl.sub.2, ZnSO.sub.4, ZrOCl.sub.2, and mixtures
and salts thereof. If selenium, selenite or selenomethionone is
present, it is at a concentration of approximately 0.002 to
approximately 0.02 mg/L. In addition, hydroxylapatite may also be
present.
[0059] It is contemplated that amino acids can be added to the
defined media. Non-limiting examples of such amino acids are
Glycine, L-Alanine, L-Alanyl-L-Glutamine, L-Glutamine/Glutamax,
L-Arginine hydrochloride, L-Asparagine-H.sub.2O, L-Aspartic acid,
L-Cysteine hydrochloride-H.sub.2O, L-Cystine 2HCl, L-Glutamic Acid,
L-Histidine hydrochloride-H.sub.2O, L-Isoleucine, L-Leucine,
L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline,
L-Hydroxyproline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine
disodium salt dihydrate, and L-Valine. In certain embodiments, the
amino acid is L-Isoleucine, L-Phenylalanine, L-Proline,
L-Hydroxyproline, L-Valine, and mixtures thereof.
[0060] It is also contemplated that the defined medium can comprise
ascorbic acid. Preferably ascorbic acid is present at an initial
concentration of approximately 1 mg/L to approximately 1000 mg/L,
or from approximately 2 mg/L to approximately 500 mg/L, or from
approximately 5 mg/L to approximately 100 mg/L, or from
approximately 10 mg/L to approximately 100 mg/L or approximately at
50 mg/L.
[0061] In addition, the compositions and methods may also comprise
other components such as serum albumin, transferrin, L-glutamine,
lipids, antibiotics, .beta.-Mercaptoethanol, vitamins, minerals,
ATP and similar components may be present. Examples of vitamins
that may be present include, but are not limited to vitamins A,
B.sub.1, B.sub.2, B.sub.3, B.sub.5, B.sub.6, B.sub.7, B.sub.9,
B.sub.12, C, D.sub.1, D.sub.2, D.sub.3, D.sub.4, D.sub.5, E,
tocotrienols, K.sub.1 and K.sub.2. One of skill in the art can
determine the optimal concentration of minerals, vitamins, ATP,
lipids, essential fatty acids, etc., for use in a given culture.
The concentration of supplements may, for example, be from about
0.001 .mu.M to about 1 mM or more. Specific examples of
concentrations at which the supplements may be provided include,
but are not limited to about 0.005 .mu.M, 0.01 .mu.M, 0.05 .mu.M,
0.1 .mu.M, 0.5 .mu.M, 1.0 .mu.M, 2.0 .mu.M, 2.5 .mu.M, 3.0M 4.0M,
5.0M, 10 .mu.M, 20 .mu.M, 100 .mu.M, etc. In one specific
embodiment, the compositions and methods comprise vitamin B.sub.6
and glutamine. In another specific embodiment, the compositions and
methods comprise vitamin C and an iron supplement. In another
specific embodiment, the compositions and methods comprise vitamin
K.sub.1 and vitamin A. In another specific embodiment, the
compositions and methods comprise vitamin D.sub.3 and ATP. In
another specific embodiment, the compositions and methods comprise
vitamin B.sub.12 and transferrin. In another specific embodiment,
the compositions and methods comprise tocotrienols and
.beta.-Mercaptoethanol. In another specific embodiment, the
compositions and methods comprise glutamine and ATP. In another
specific embodiment, the compositions and methods comprise an
omega-3 fatty acid and glutamine. In another specific embodiment,
the compositions and methods comprise an omega-6 fatty acid and
vitamin B.sub.1. In another specific embodiment, the compositions
and methods comprise .alpha.-linolenic acid and B.sub.2.
[0062] The compositions of the present invention are essentially
serum free. As used herein, "essentially serum free" refers to the
absence of serum in the solutions of the present invention. Serum
is not an essential ingredient to the compositions and methods of
the present invention. Thus, the presence of serum in any of the
compositions should only be attributable to impurities, e.g., from
the starting materials or residual serum from the primary cell
culture. For example, essentially serum free medium or environment
can contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% serum
wherein the presently improved bioactive maintenance capacity of
the medium or environment is still observed. In a specific
embodiment of the present invention, the essentially serum free
composition does not contain serum or serum replacement, or only
contains trace amounts of serum or serum replacement from the
isolation of components of the serum or serum replacement that are
added to the defined media.
[0063] The compositions and methods of the present invention also
comprise a means for stimulating ErbB2 tyrosine kinase activity
within differentiable cells. In one specific embodiment, the
compositions and methods of the present invention comprise the
presence of at least one ErbB3 ligand. Typically, an ErbB3 ligand
will bind the ErbB3 receptor and dimerize with the ErbB2 receptor.
The ErbB2 receptor is, in turn, generally responsible for
intracellular tyro sine kinase activity within the differentiable
cell.
[0064] As used herein, "ErbB3 ligand" refers to a ligand that binds
to ErbB3, which in turn dimerizes to ErbB2, thus activating the
tyrosine kinase activity of the ErbB2 portion of the ErbB2/ErbB3
heterodimeric receptor. Non-limiting examples of ErbB3 ligands
include Neuregulin-1; splice variants and isoforms of Neuregulin-1,
including but not limited to HRG-.beta., HRG-.alpha., Neu
Differentiation Factor (NDF), Acetylcholine Receptor-Inducing
Activity (ARIA), Glial Growth Factor 2 (GGF2), and Sensory And
Motor Neuron-Derived Factor (SMDF); Neuregulin-2; splice variants
and isoforms of Neuregulin-2, including but not limited to
NRG2-.beta.; Epiregulin; and Biregulin.
[0065] In one embodiment, the means for stimulating ErbB2-directed
tyrosine kinase activity comprise at least one ErbB3 ligand that is
selected from the group consisting of Neuregulin-1,
Heregulin-.beta. (HRG-.beta.), Heregulin-.alpha. (HRG-.alpha.), Neu
differentiation factor (NDF), acetylcholine receptor-inducing
activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived
factor (SMDF), Neuregulin-2, Neuregulin-2.beta. (NRG2-.beta.),
Epiregulin, Biregulin and variants and functional fragments
thereof. In another specific embodiment, the compositions and
methods of the present invention comprise more than one means for
stimulating ErbB2-directed tyrosine kinase activity, such as, but
not limited to, using more than one ErbB3 ligand.
[0066] In a more specific embodiment of the compositions and
methods of the present invention, the ErbB3 ligand is HRG-.beta. or
a variant or functional fragment thereof. In one embodiment, the
species from which the culture additive protein, polypeptide or
variant or functional fragment thereof derives is the same as the
species of cells that are cultured. For example, if mouse ES cells
are cultured, an HRG-.beta. with an amino acid sequence that is
identical to the mus musculus HRG-.beta. sequence can be used as an
additive in culture and is considered to be "of the same species."
In other embodiments, the species from which the biological
additive derives is different from the cells being cultures. For
example, if mouse ES cells are cultured, an HRG-.beta. with an
amino acid sequence that is identical to the human HRG-.beta.
sequence from can be used as an additive in culture and is
considered to be "of different species."
[0067] As used herein, a "functional fragment" is a fragment or
splice variant of a full length polypeptide that exerts a similar
physiological or cellular effect as the full length polypeptide.
The biological effect of the functional fragment need not be
identical in scope or strength as the full-length polypeptide, so
long as a similar physiological or cellular effect is seen. For
example, a functional fragment of HRG-.beta. can detectably
stimulate ErbB2-directed tyrosine kinase.
[0068] As used herein, the term "variant" includes chimeric or
fusion polypeptides, homologs, analogs, orthologs, and paralogs. In
addition, a variant of a reference protein or polypeptide is a
protein or polypeptide whose amino acid sequence is at least about
80% identical to the reference protein or polypeptide. In specific
embodiments, the variant is at least about 85%, 90%, 95%, 95%, 97%,
98%, 99% or even 100% identical to the reference protein or
polypeptide. As used herein, the terms "correspond(s) to" and
"corresponding to," as they relate to sequence alignment, are
intended to mean enumerated positions within the reference protein
or polypeptide, e.g., wild-type human or mouse neuregulin-1, and
those positions in the modified protein or polypeptide that align
with the positions on the reference protein or polypeptide. Thus,
when the amino acid sequence of a subject protein or polypeptide is
aligned with the amino acid sequence of a reference protein or
polypeptide, the sequence that "corresponds to" certain enumerated
positions of the reference protein or polypeptide sequence are
those that align with these positions of the reference sequence,
but are not necessarily in these exact numerical positions of the
reference sequence. Methods for aligning sequences for determining
corresponding amino acids between sequences are described
below.
[0069] A polypeptide having an amino acid sequence at least, for
example, about 95% "identical" to a reference an amino acid
sequence encoding, for example TGF-.beta., is understood to mean
that the amino acid sequence of the polypeptide is identical to the
reference sequence except that the amino acid sequence may include
up to about five modifications per each 100 amino acids of the
reference amino acid sequence encoding the reference TGF-.beta.. In
other words, to obtain a peptide having an amino acid sequence at
least about 95% identical to a reference amino acid sequence, up to
about 5% of the amino acid residues of the reference sequence may
be deleted or substituted with another amino acid or a number of
amino acids up to about 5% of the total amino acids in the
reference sequence may be inserted into the reference sequence.
These modifications of the reference sequence may occur at the
N-terminus or C-terminus positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among amino acids in the reference sequence or
in one or more contiguous groups within the reference sequence.
[0070] As used herein, "identity" is a measure of the identity of
nucleotide sequences or amino acid sequences compared to a
reference nucleotide or amino acid sequence. In general, the
sequences are aligned so that the highest order match is obtained.
"Identity" per se has an art-recognized meaning and can be
calculated using published techniques. (See, e.g., Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York (1988); Biocomputing: Informatics And Genome Projects, Smith,
D. W., ed., Academic Press, New York (1993); Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis
In Molecular Biology, Academic Press (1987); and Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York (1991)). While there exist several methods to measure identity
between two polynucleotide or polypeptide sequences, the term
"identity" is well known to skilled artisans (Carillo, H. &
Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly
employed to determine identity or similarity between two sequences
include, but are not limited to, those disclosed in Guide to Huge
Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994)
and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073
(1988). Computer programs may also contain methods and algorithms
that calculate identity and similarity. Examples of computer
program methods to determine identity and similarity between two
sequences include, but are not limited to, GCG program package
(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)),
BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol
215:403 (1990)) and FASTDB. Examples of methods to determine
identity and similarity are discussed in Michaels, G. and Garian,
R., Current Protocols in Protein Science, Vol 1, John Wiley &
Sons, Inc. (2000), which is incorporated by reference. In one
embodiment of the present invention, the algorithm used to
determine identity between two or more polypeptides is BLASTP.
[0071] In another embodiment of the present invention, the
algorithm used to determine identity between two or more
polypeptides is FASTDB, which is based upon the algorithm of
Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated
by reference). In a FASTDB sequence alignment, the query and
subject sequences are amino sequences. The result of sequence
alignment is in percent identity. Parameters that may be used in a
FASTDB alignment of amino acid sequences to calculate percent
identity include, but are not limited to: Matrix=PAM, k-tuple=2,
Mismatch Penalty=1, Joining Penalty=20, Randomization Group
Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05,
Window Size=500 or the length of the subject amino sequence,
whichever is shorter.
[0072] If the subject sequence is shorter or longer than the query
sequence because of N-terminus or C-terminus additions or
deletions, not because of internal additions or deletions, a manual
correction can be made, because the FASTDB program does not account
for N-terminus and C-terminus truncations or additions of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are N- and C-terminus to
the reference sequence that are not matched/aligned, as a percent
of the total bases of the query sequence. The results of the FASTDB
sequence alignment determine matching/alignment. The alignment
percentage is then subtracted from the percent identity, calculated
by the above FASTDB program using the specified parameters, to
arrive at a final percent identity score. This corrected score can
be used for the purposes of determining how alignments "correspond"
to each other, as well as percentage identity. Residues of the
query (subject) sequences or the reference sequence that extend
past the N- or C-termini of the reference or subject sequence,
respectively, may be considered for the purposes of manually
adjusting the percent identity score. That is, residues that are
not matched/aligned with the N- or C-termini of the comparison
sequence may be counted when manually adjusting the percent
identity score or alignment numbering.
[0073] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue reference sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
match/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues
at the N- and C-termini not matched/total number of residues in the
query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90
residues were perfectly matched the final percent identity would be
90%. In another example, a 90 residue subject sequence is compared
with a 100 reference sequence. This time the deletions are internal
deletions so there are no residues at the N- or C-termini of the
subject sequence which are not matched/aligned with the query. In
this case the percent identity calculated by FASTDB is not manually
corrected.
[0074] The invention also provides chimeric or fusion polypeptides.
As used herein, a "chimeric polypeptide" or "fusion polypeptide"
comprises at least a portion of a member of the reference
polypeptide operatively linked to a second, different polypeptide.
The second polypeptide has an amino acid sequence corresponding to
a polypeptide which is not substantially identical to the reference
polypeptide, and which is derived from the same or a different
organism. With respect to the fusion polypeptide, the term
"operatively linked" is intended to indicate that the reference
polypeptide and the second polypeptide are fused to each other so
that both sequences fulfill the proposed function attributed to the
sequence used. The second polypeptide can be fused to the
N-terminus or C-terminus of the reference polypeptide. For example,
in one embodiment, the fusion polypeptide is a GST-IGF-1 fusion
polypeptide in which an IGF-1 sequence is fused to the C-terminus
of the GST sequences. Such fusion polypeptides can facilitate the
purification of recombinant polypeptides. In another embodiment,
the fusion polypeptide can contain a heterologous signal sequence
at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression and/or secretion of a polypeptide can be
increased through use of a heterologous signal sequence.
[0075] In addition to fragments and fusion polypeptides, the
present invention includes homologs and analogs of naturally
occurring polypeptides. "Homologs" are defined herein as two
nucleic acids or polypeptides that have similar, or "identical,"
nucleotide or amino acid sequences, respectively. Homologs include
allelic variants, orthologs, paralogs, agonists, and antagonists as
defined hereafter. The term "homolog" further encompasses nucleic
acid molecules that differ from a reference nucleotide sequence due
to degeneracy of the genetic code and thus encode the same
polypeptide as that encoded by the reference nucleotide sequence.
As used herein, "naturally occurring" refers to a nucleic or amino
acid sequence that occurs in nature.
[0076] An agonist of a polypeptide can retain substantially the
same, or a subset, of the biological activities of the polypeptide.
An antagonist of a polypeptide can inhibit one or more of the
activities of the naturally occurring form of the polypeptide.
[0077] In another more specific embodiment of the compositions and
methods of the present invention, the ErbB3 ligand is HRG-.beta. or
a variant or a functional fragment thereof. Additional,
non-limiting examples of ErbB3 ligands are disclosed in U.S. Pat.
Nos. 6,136,558, 6,387,638, and 7,063,961, which are incorporated by
reference.
[0078] Heregulins are generally classified into two major types,
alpha and beta, based on two variant EGF-like domains that differ
in their C-terminal portions. These EGF-like domains, however, are
identical in the spacing of six cysteine residues contained
therein. Based on an amino acid sequence comparison, Holmes et al.
found that between the first and sixth cysteines in the EGF-like
domain, HRGs were 45% similar to heparin-binding EGF-like growth
factor (HB-EGF), 35% identical to amphiregulin (AR), 32% identical
to TGF-.alpha., and 27% identical to EGF.
[0079] The 44 kDa neu differentiation factor (NDF) is the rat
equivalent of human HRG. Like the HRG polypeptides, NDF has an
immunoglobulin (Ig) homology domain followed by an EGF-like domain
and lacks a N-terminal signal peptide. Presently, there are at
least six distinct fibroblastic pro-NDFs, classified as either
alpha or beta polypeptides, based on the sequences of the EGF-like
domains. Isoforms 1 to 4 are characterized on the basis of a
variable stretch between the EGF-like domain and transmembrane
domain. Thus it appears that different NDF isoforms are generated
by alternative splicing and may perform distinct tissue-specific
functions. See EP 505 148; WO 93/22424; and WO 94/28133, which are
incorporated by reference.
[0080] In one embodiment of the present invention, the compositions
and methods are free of exogenous insulin and insulin substitutes.
The phrase "exogenous insulin or insulin substitutes" is used
herein to indicate insulin or insulin substitutes that is/are not
intentionally added to the compositions or methods of the present
invention. Thus, in certain embodiments of the present invention,
the methods and compositions are free of insulin or insulin
substitutes that are intentionally supplied. The compositions or
methods may, however, not necessarily be free of endogenous
insulin. As used herein, "endogenous insulin" indicates that the
cultured cells may be producing insulin of their own accord when
cultured according to the methods of the present invention.
Endogenous insulin also may be used to indicate residual impurities
from the primary cell culture or impurities from the starting
materials. In specific examples, the compositions and methods of
the present contain less than 50, 45, 40, 35, 30, 25, 20, 15, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 .mu.g/ml of insulin.
[0081] As used herein, the term "insulin" refers to the protein, or
variant or fragment thereof that binds to the insulin receptor in
normal physiological concentrations and can induce signaling
through the insulin receptor. The term "insulin" encompasses a
protein having the polypeptide sequence of native human insulin, or
of other mammalian insulin, or of any homologs or variants to these
sequences. Additionally, the term insulin encompasses polypeptide
fragments that are capable of binding to the insulin receptor to
induce signaling through the insulin receptor. The term "insulin
substitute" refers to any zinc containing compound that may be used
in place of insulin to give substantially similar results as
insulin. Examples of insulin substitutes include, but are not
limited to zinc chloride, zinc nitrate, zinc bromide, and zinc
sulfate.
[0082] To be clear, insulin-like growth factors are not insulin
substitutes or homologs of insulin, as contemplated in the present
invention. Accordingly, in another specific embodiment, the
compositions and methods of the present invention comprise the use
of at least one insulin-like growth factor (IGF) or a variant or a
functional fragment thereof. In another embodiment, the
compositions and methods of the present invention are free of any
exogenous insulin-like growth factors (IGFs). In specific
embodiments, the compositions and methods of the present invention
contain less than 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7,
6, 5, 4, 3, 2, or 1 ng/ml of IGF-1.
[0083] As used herein, the term "activator of IGF-1R" refers to
mitogens that play a pivotal role in regulating cell proliferation,
differentiation, and apoptosis. The effects of an activator of
IGF-1R are typically mediated through IGF-1R, although they can be
mediated through other receptors. The IGF-1R is also involved in
cell transformation induced by tumor virus proteins and oncogene
products, and the interaction is regulated by a group of specific
binding proteins (IGFBPs). In addition, a large group of IGFBP
proteases hydrolyze IGFBPs, resulting in the release of bound IGFs
that then resume their ability to interact with IGF-IR. For the
purpose of this invention, the ligands, the receptors, the binding
proteins, and the proteases are all considered to be activators of
IGF-1R. In one embodiment, the activator of IGF-1R is IGF-1, or
IGF-2. In a further embodiment, the activators of IGF-1R is an
IGF-1 analog. Non-limiting examples of IGF-1 analogs include
LongR3-IGF1, Des(1-3)IGF-1, [Arg.sup.3]IGF-1, [Ala.sup.31]IFG-1,
Des(2,3)[Ala.sup.31]IGF-1, [Leu.sup.24]IGF1,
Des(2,3)[Leu.sup.24]IGF-1, [Leu.sup.60]IGF-1,
[Ala.sup.31][Leu.sup.60]IGF-1, [Leu.sup.24][Ala.sup.31]IGF-1, and
combinations thereof. In a further embodiment, the IFG-1 analog is
LongR3-IGF1, which is a recombinant analog of human insulin growth
factor-1. It is contemplated that LongR3-IGF1 is initially present
at a concentration of approximately 1 ng/ml to approximately 1000
ng/ml, more preferably approximately 5 ng/ml to approximately 500
ng/ml, more preferably approximately 50 ng/ml to approximately 500
ng/ml, more preferably approximately 100 ng/ml to approximately 300
ng/ml, or at a concentration of approximately 100 ng/ml.
[0084] In certain embodiments, the compositions and methods of the
present invention comprise transforming growth factor beta
(TGF-.beta.) or a TGF-.beta. family member or variants or
functional fragments thereof. As used herein, the term "member of
the TGF-.beta. family" or the like refers to growth factors that
are generally characterized by one of skill in the art as belonging
to the TGF-13 family, either due to homology with known members of
the TGF-13 family, or due to similarity in function with known
members of the TGF-13 family. In particular embodiments of the
invention, if the member of the TGF-13 family is present, the
TGF-13 family member of variant or functional fragment thereof
activates SMAD 2 or 3. In certain embodiments, the member of the
TGF-13 family is selected from the group consisting of Nodal,
Activin A, Activin B, TGF-13, bone morphogenic protein-2 (BMP2) and
bone morphogenic protein-4 (BMP4). In one embodiment, the member of
the TGF-.beta. family is Activin A.
[0085] It is contemplated that if Nodal is present, it is initially
present at a concentration of approximately 0.1 ng/ml to
approximately 2000 ng/ml, more preferably approximately 1 ng/ml to
approximately 1000 ng/ml, more preferably approximately 10 ng/ml to
approximately 750 ng/ml, or more preferably approximately 25 ng/ml
to approximately 500 ng/ml. It is contemplated that if used,
Activin A is initially present at a concentration of approximately
0.01 ng/ml to approximately 1000 ng/ml, more preferably
approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably
approximately 0.1 ng/ml to approximately 25 ng/ml, or most
preferably at a concentration of approximately 10 ng/ml. It is
contemplated that if present, TGF-.beta. is initially present at a
concentration of approximately 0.01 ng/ml to approximately 100
ng/ml, more preferably approximately 0.1 ng/ml to approximately 50
ng/ml, or more preferably approximately 0.1 ng/ml to approximately
20 ng/ml.
[0086] In additional embodiments of the present invention, the
compositions and methods of the present invention are free of
activators of FGF receptors. There are currently at least 22 known
members of the family of fibroblast growth factors, with these
factors binding to one of at least one of four FGF receptors. As
used herein, the term "activator of an FGF receptor" refers to
growth factors that are generally characterized by one of skill in
the art as belonging to the FGF family, either due to homology with
known members of the FGF family, or due to similarity in function
with known members of the FGF family. In certain embodiments, the
activator of an FGF receptor is an FGF, such as, but not limited to
.alpha.-FGF and FGF2. In particular embodiments, the compositions
and methods are free of exogenous FGF2. The phrase "exogenous FGF2"
is used herein to indicate fibroblast growth factor 2, i.e., basic
FGF, that is not intentionally added to the compositions or methods
of the present invention. Thus, in certain embodiments of the
present invention, the methods and compositions are free of
intentionally supplied FGF2. The compositions or methods may,
however, not necessarily be free of endogenous FGF2. As used
herein, "endogenous FGF2" indicates that the cultured cells may be
producing FGF2 of their own accord when cultured according to the
methods of the present invention. "Endogenous FGF2" also may be
used to indicate residual impurities from the primary cell culture
or impurities from the starting materials. In specific examples,
the compositions and methods of the present contain less than 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml of FGF2.
[0087] It is contemplated, however, that the compositions and
methods of the invention can include at least one activator of an
FGF receptor, including any of the FGF polypeptides, functional
fragments thereof or variants thereof. It is contemplated that if
FGF2 is present, it is initially present at a concentration of
approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably
approximately 0.5 ng/ml to approximately 50 ng/ml, more preferably
approximately 1 ng/ml to approximately 25 ng/ml, more preferably
approximately 1 ng/ml to approximately 12 ng/ml, or most preferably
at a concentration of approximately 8 ng/ml. In another specific
embodiment, the compositions and methods of the invention can
include at least one activator of an FGF receptor, other than FGF2.
For example, the compositions and methods of the present invention
may comprise at least one of FGF-7, FGF-10 or FGF-22 or variants or
functional fragments thereof. In specific embodiments, a
combination of at least two of FGF-7, FGF-10 and FGF-22, or
variants or functional fragments thereof, are present. In another
embodiment, all three of FGF-7, FGF-10 and FGF-22, or variants or
functional fragments thereof, are present. It is contemplated that
if any of FGF-7, FGF-10 or FGF-22 or variants or functional
fragments are present, each is initially present at a concentration
of approximately 0.1 ng/ml to approximately 100 ng/ml, more
specifically from approximately 0.5 ng/ml to approximately 50
ng/ml, more specifically from approximately 1 ng/ml to
approximately 25 ng/ml, more specifically from approximately 1
ng/ml to approximately 12 ng/ml, or most specifically at a
concentration of approximately 8 ng/ml.
[0088] In additional certain embodiments, the compositions and
methods of the present invention comprise serum albumin (SA). In
specific embodiments, the SA is either bovine SA (BSA) or human SA
(HAS). In still more specific embodiments, the concentration of the
SA is more than about 0.2%, volume to volume (v/v), but less than
about 10% v/v. In even more specific embodiments, the concentration
of SA is more than about 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,
1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%,
3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%,
5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%,
7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%
and 9.8% (v/v).
[0089] In additional embodiments, the compositions and methods
comprise at least one insoluble substrate. For example, the
differentiable cells may be placed on a cell culture surface that
comprises such compounds as, but is not limited to, polystyrene,
polypropylene. The surface may, in turn, be coated with an
insoluble substrate. In specific embodiments, the insoluble
substrate is selected from the group consisting of a collagen, a
fibronectin and fragments or variants thereof. Other examples of
insoluble substrates include, but are not limited to, fibrin,
elastin, fibronectins, laminins and nidogens.
[0090] Accordingly, the cell culture environments and methods of
the present invention comprise plating the cells in an adherent
culture. As used herein, the terms "plated" and "plating" refer to
any process that allows a cell to be grown in adherent culture. As
used herein, the term "adherent culture" refers to a cell culture
system whereby cells are cultured on a solid surface, which may in
turn be coated with an insoluble substrate that may in turn be
coated with another surface coat of a substrate, such as those
listed below, or any other chemical or biological material that
allows the cells to proliferate or be stabilized in culture. The
cells may or may not tightly adhere to the solid surface or to the
substrate. The substrate for the adherent culture may comprise any
one or combination of polyornithine, laminin, poly-lysine, purified
collagen, gelatin, fibronectin, tenascin, vitronectin, entactin,
heparin sulfate proteoglycans, poly glycolytic acid (PGA), poly
lactic acid (PLA), and poly lactic-glycolic acid (PLGA).
Furthermore, the substrate for the adherent culture may comprise
the matrix laid down by a feeder layer, or laid down by the
pluripotent human cell or cell culture. As used herein, the term
"extracellular matrix" encompasses solid substrates such as but not
limited to those described above, as well as the matrix laid down
by a feeder cell layer or by the pluripotent human cell or cell
culture. In one embodiment, the cells are plated on
MATRIGEL.TM.-coated plates. In another embodiment, the cells are
plated on fibronectin-coated plates. In certain embodiments, if the
cells are plated on fibronectin, the plates are prepared by coating
with 10 .mu.g/ml human plasma fibronectin (Invitrogen, #33016-015),
diluted in tissue grade water, for 2-3 hours at room temperature.
In another embodiment, serum can be placed in the medium for up to
24 hours to allow cells to plate to the plastic. If using serum to
promote the attachment of the cells, the media is then removed and
the compositions, which are essentially serum-free, are added to
the plated cells.
[0091] The compositions and methods of the present invention
contemplate that the differentiable cells are cultured in
conditions that are essentially free of a feeder cell or feeder
layer. As used herein, a "feeder cell" is a cell that grows in
vitro, that is co-cultured with a target cell and stabilizes the
target cell in its current state of differentiation. As used
herein, a "feeder cell layer" can be used interchangeably with the
term "feeder cell." As used herein, the term "essentially free of a
feeder cell" refers to tissue culture conditions that do not
contain feeder cells, or that contain a de minimus number of feeder
cells. By "de minimus", it is meant that number of feeder cells
that are carried over to the instant culture conditions from
previous culture conditions where the differentiable cells may have
been cultured on feeder cells. In one embodiment of the above
method, conditioned medium is obtained from a feeder cell that
stabilizes the target cell in its current state of differentiation.
In another embodiment, the defined medium is a non-conditioned
medium, which is a medium that is not obtained from a feeder
cell.
[0092] As used herein, the term "stabilize," when used in reference
to the differentiation state of a cell or culture of cells,
indicates that the cells will continue to proliferate over multiple
passages in culture, and preferably indefinitely in culture, where
most, if not all, of the cells in the culture are of the same
differentiation state. In addition, when the stabilized cells
divide, the division typically yield cells of the same cell type or
yield cells of the same differentiation state. A stabilized cell or
cell population in general, does not further differentiate or
de-differentiate if the cell culture conditions are not altered,
and the cells continue to be passaged and are not overgrown. In one
embodiment, the cell that is stabilized is capable of proliferation
in the stable state indefinitely, or for at least more than 2
passages. In a more specific embodiment, the cells are stable for
more than 3 passages, 4 passages, 5 passages, 6 passages, 7
passages, 8 passages, 9 passages, more than 10 passages, more than
15 passages, more than 20 passages, more than 25 passages, or more
than 30 passages. In one embodiment, the cell is stable for greater
than approximately 1 month, 2 months, 3 months, 4 months, 5 months,
6 months, 7 months, 8 months, 9 months, 10 months, or 11 months of
continuous passaging. In another embodiment, the cell is stable for
greater than approximately 1 year of continuous passaging. In one
embodiment, stem cells are maintained in culture in a pluripotent
state by routine passage in the defined medium until it is desired
that they be differentiated. As used herein, the term "proliferate"
refers to an increase in the number cells in a cell culture.
[0093] In certain embodiments, the compositions and methods
comprise an inactivator of BMP signaling. As used herein, an
"inactivator of BMP signaling" refers to an agent that antagonizes
the activity of one or more BMP proteins or any of their upstream
or downstream signaling components through any of its possible
signaling pathways. The compound(s) used to inactivate BMP
signaling can be any compound known in the art, or later
discovered. Non-limiting examples of inactivators of BMP signaling
include dominant-negative, truncated BMP receptor, soluble BMP
receptors, BMP receptor-Fc chimeras, noggin, follistatin, chordin,
gremlin, cerberus/DAN family proteins, ventropin, high dose
activin, and amnionless.
[0094] In certain embodiments, the compositions and methods can
comprise at least one hormone, cytokine, adipokine, growth hormone
or variant or functional fragment thereof. It is currently
contemplated that in certain embodiments, the growth hormone
present in the defined medium will be of the same species as the
differentiable cells that are cultured with the defined media.
Thus, for example, if a human cell is cultured, the growth hormone
is human growth hormone. The use of growth hormone that is from a
species different than the cultured cells is also contemplated.
Preferably the hormone, cytokine, adipokine and/or growth hormone
is present at an initial concentration of approximately 0.001 ng/ml
to approximately 1000 ng/ml, more preferably approximately 0.001
ng/ml to approximately 250 ng/ml, or more preferably approximately
0.01 ng/ml to approximately 150 ng/ml.
[0095] Examples of cytokines and adipokines that may be included in
the compositions and methods of the present invention include, but
are not limited to, the four .alpha.-helix bundle family of
cytokines, the interleukin-1 (IL-1) family of cytokines, the IL-17
family of cytokines and the chemokine family of cytokines. Of
course, the invention contemplates members and subclasses of each
of these families of cytokines, such as, but not limited to, the CC
chemokines, the CXC chemokines, the C chemokines and the CX.sub.3C
chemokines, interferons, interleukins, lymphotoxins, c-kit ligand,
granulocyte-macrophage colony-stimulating factor (GM-CSF),
monocyte-macrophage colony-stimulating factor (M-CSF), granulocyte
colony-stimulating factor (G-CSF), leptin, adiponectin, resistin,
plasminogen activator inhibitor-1 (PAI-1), tumor necrosis
factor-alpha (TNF.alpha.), tumor necrosis factor-beta (TNF.beta.),
leukemia inhibitory factor, visfatin, retinol binding protein 4
(RBP4), erythropoietin (EPO), thrombopoietin (THPO). Of course, one
of skill in the art will understand that the invention contemplates
variants or functional fragments of the above-listed factors.
[0096] The present invention relates to methods of culturing
differentiable cells, with the methods comprising plating
differentiable cells on a cell culture surface, providing a basal
salt nutrient solution to the cells and providing a means for
stimulating ErbB2-directed tyrosine kinase activity in the
cells.
[0097] In one embodiment, differentiable cells are contacted with
the at least one of the compositions of the invention in the
absence of serum or serum replacement, and in the absence of a
feeder cell layer, such that the cells are maintained in an
undifferentiated state for at least one month. Pluripotency can be
determined through characterization of the cells with respect to
surface markers, transcriptional markers, karyotype, and ability to
differentiate to cells of the three germ layers. These
characteristics are well known to those of ordinary skill in the
art.
[0098] It is contemplated that the differentiable cells can be
passaged using enzymatic, non-enzymatic, or manual dissociation
methods prior to and/or after contact with the defined medium of
the invention. Non-limiting examples of enzymatic dissociation
methods include the use of proteases such as trypsin, collagenase,
dispase, and ACCUTASE.TM.. In one embodiment, ACCUTASE.TM. is used
to passage the contacted cells. When enzymatic passaging methods
are used, the resultant culture can comprise a mixture of singlets,
doublets, triplets, and clumps of cells that vary in size depending
on the enzyme used. A non-limiting example of a non-enzymatic
dissociation method is a cell dispersal buffer. Manual passaging
techniques have been well described in the art, such as in Schulz
et al., 2004 Stem Cells, 22(7):1218-38. The choice of passaging
method is influenced by the choice of extracellular matrix, and is
easily determined by one of ordinary skill in the art.
[0099] In one specific embodiment, methods of culturing
differentiable cells comprise providing a dissociation solution to
a layer of stem cells that are contained in a culture chamber prior
to dissociation, where the dissociation breaks apart the layer of
cells into single cells. After dissociation, the single cells are
placed into a new tissue culture chamber with a stem cell culture
solution, wherein the stem cell culture solution comprises a basal
salt nutrient solution and an ErbB3 ligand. Once cultured, the
single stem cells are placed in conditions that permit growth and
division of the single cells.
[0100] The disaggregation solution used in the methods of the
present invention can be any disaggregation solution capable of
breaking apart or disaggregating the cells into single cells,
without causing extensive toxicity to the cells. Examples of
disaggregation solutions include, but are not limited to, trypsin,
ACCUTASE.TM., 0.25% Trypsin/EDTA, TrypLE, or VERSENE.TM. (EDTA) and
trypsin. The methods of the present invention need not result in
every cell of the confluent layer being disaggregated into single
cells, provided that at least a few single cells are disaggregated
and capable of being re-cultured.
[0101] Differentiable cells may also be utilized to screen for
molecules or factors that influence their plasticity or other
characterisitics. For example, differentiable cells could be used
to identify agents that induce apoptosis, differentiation or
proliferation, as well as similar effects in differentiated
lineages that have been generated from the differentiable
cells.
[0102] Because the compositions and methods of the present
invention allow for single cell passaging, differentiable cells
have been successfully cultured in high-throughput settings, such
as, but not limited to, 96-well plates and 384-well plates. FIG. 16
shows the morphology and alkaline phosphatase staining of BG02
cells that were cultured in DC-HAIF in both a 96-well and 384-well
plate, using the methods described herein. Briefly, hESCs cells
that were split, using ACCUTASE.TM., and plated in 96-well and
384-well plates and cultured showed a similar plating efficiency as
what is observed using other culture dishes. In addition, the cells
formed colonies, and were expanded successfully over 5 days in the
smaller environments. These smaller cultures remained
morphologically undifferentiated, and stained uniformly positive
for alkaline phosphatase, a marker of undifferentiated cells.
Furthermore, hESCs could also be grown in 96-well culture devices
(not shown) that provide real-time measurements of impedance, which
can be used to measure cell proliferation and viability using the
RT-CES.TM. methods from ACEA Biosciences, Inc. (www.aceabio.com).
Such an approach would enable a label-free identification and
quantitation of subtle or immediate effects on differentiable
cells, as well as measurements of proliferation, apoptosis and
changes to morphology, in real time.
[0103] The compositions and methods of the invention may contain
virtually any combination of the components set out above or
described elsewhere herein, provided the compositions and methods
comprise a basal salt nutrient solution and a means for stimulating
ErbB2 directed tyrosine kinase activity. As one skilled in the art
would recognize, the components of the compositions and methods of
the invention will vary according to the protocol design.
Accordingly, one embodiment of the present invention relates to
culturing differentiable cells in 96-well plates and/or 384-well
plates. Indeed, using the methods and compositions of the present
invention, the cell culture chamber, i.e., the culture dish, is no
longer limited to specific dimensions. Thus, the methods of the
present invention is not limited to specific culture chamber
dimensions.
[0104] The compositions and methods described herein have several
useful features. For example, the compositions and methods
described herein are useful for modeling the early stages of human
development. Furthermore, the compositions and methods described
herein can also serve for therapeutic intervention in disease
states, such as neurodegenerative disorders, diabetes mellitus or
renal failure, such as by the development of pure tissue or cell
type.
[0105] The cell types that differentiate from differentiable cells
have several uses in various fields of research and development
including but not limited to drug discovery, drug development and
testing, toxicology, production of cells for therapeutic purposes
as well as basic science research. These cell types express
molecules that are of interest in a wide range of research fields.
These include the molecules known to be required for the
functioning of the various cell types as described in standard
reference texts. These molecules include, but are not limited to,
cytokines, growth factors, cytokine receptors, extracellular
matrix, transcription factors, secreted polypeptides and other
molecules, and growth factor receptors.
[0106] It is contemplated that the differentiable cells of the
invention can be differentiated through contact with a cell
differentiation environment. As used herein, the term "cell
differentiation environment" refers to a cell culture condition
wherein the differentiable cells are induced to differentiate, or
are induced to become a human cell culture enriched in
differentiated cells. Preferably, the differentiated cell lineage
induced by the growth factor will be homogeneous in nature. The
term "homogeneous," refers to a population that contains more than
approximately 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired cell
lineage.
[0107] A cell differentiating medium or environment may be utilized
to partially, terminally, or reversibly differentiate the
differentiable cells of the present invention. In accordance with
the invention the medium of the cell differentiation environment
may contain a variety of components including, for example, KODMEM
medium (Knockout Dulbecco's Modified Eagle's Medium), DMEM, Ham's
F12 medium, FBS (fetal bovine serum), FGF2 (fibroblast growth
factor 2), KSR or hLIF (human leukemia inhibitory factor). The cell
differentiation environment can also contain supplements such as
L-Glutamine, NEAA (non-essential amino acids), P/S
(penicillin/streptomycin), N2, B27 and .beta.-mercaptoethanol
(.beta.-ME). It is contemplated that additional factors may be
added to the cell differentiation environment, including, but not
limited to, fibronectin, laminin, heparin, heparin sulfate,
retinoic acid, members of the epidermal growth factor family
(EGFs), members of the fibroblast growth factor family (FGFs)
including FGF2 and/or FGF8, members of the platelet derived growth
factor family (PDGFs), transforming growth factor (TGF)/bone
morphogenetic protein (BMP)/growth and differentiation factor (GDF)
factor family antagonists including but not limited to noggin,
follistatin, chordin, gremlin, cerberus/DAN family proteins,
ventropin, high dose activin, and amnionless or variants or
functional fragments thereof TGF/BMP/GDF antagonists could also be
added in the form of TGF/BMP/GDF receptor-Fc chimeras. Other
factors that may be added include molecules that can activate or
inactivate signaling through Notch receptor family, including but
not limited to proteins of the Delta-like and Jagged families as
well as inhibitors of Notch processing or cleavage, or variants or
functional fragments thereof. Other growth factors may include
members of the insulin like growth factor family (IGF), insulin,
the wingless related (WNT) factor family, and the hedgehog factor
family or variants or functional fragments thereof. Additional
factors may be added to promote mesendoderm stem/progenitor,
endoderm stem/progenitor, mesoderm stem/progenitor, or definitive
endoderm stem/progenitor proliferation and survival as well as
survival and differentiation of derivatives of these
progenitors.
[0108] The compositions described herein are useful for the
screening of test compounds to determine whether a test compound
modulates pluripotency, proliferation, and/or differentiation of
differentiable cells. Pluripotency, proliferation and/or
differentiation of differentiable cells can be readily ascertained
by one of ordinary skill in the art. Non-limiting methods include
examining cell morphology, the expression of various markers,
teratoma formation, cell counts and measurements of impedance.
[0109] The progression of the differentiable cells to the desired
cell lineage, or its maintenance in an undifferentiated state can
be monitored by quantitating expression of marker genes
characteristic of the desired cell lineage as well as the lack of
expression of marker genes characteristic of differentiable cell
types. One method of quantitating gene expression of such marker
genes is through the use of quantitative PCR (Q-PCR). Methods of
performing Q-PCR are well known in the art. Other methods that are
known in the art can also be used to quantitate marker gene
expression. Marker gene expression can be detected by using
antibodies specific for the marker gene of interest.
[0110] In certain embodiments, the screening method encompasses
methods of identifying a compound capable of modulating
pluripotency, proliferation and/or differentiation of a
differentiable cell, comprising (a) providing a differentiable
cell; (b) culturing the cell in a composition comprising a basal
salt nutrient solution and an ErbB3 ligand, wherein the composition
is essentially serum free; (c) contacting the cell with a test
compound; and determining whether an increase or decrease in
pluripotency, proliferation and/or differentiation occurs in the
cell contacted with the compound, said increase being an indication
that the compound modulates pluripotency, proliferation and/or
differentiation. In certain embodiments, the ErbB3 ligand is
HRG-.beta.. In other embodiments, the ErbB3 ligand can be
substituted with a test compound, to determine the effects of the
test compound. For example, the effects on pluripotency,
proliferation and/or differentiation that occurs with the test
compound can be compared to the effects on pluripotency,
proliferation and/or differentiation that occurs with the ErbB3
ligand to determine the effects of the test compound on the
differentiable cells. It is contemplated that any of the
compositions described herein can be used in the screening methods
of the present invention.
[0111] In yet another embodiment, the cells can be cultured in the
absence of an ErbB3 ligand (ErbB2-directed tyrosine kinase
activity) to determine the effects of the absence of an ErbB3
ligand (ErbB2-directed tyrosine kinase activity) on the cells.
[0112] Using the methods described herein, compositions comprising
the desired cell lineage that are substantially free of other cell
types can be produced. Alternatively, compositions comprising
mixtures of the differentiable cells and the desired cell lineage
can also be produced.
[0113] In some embodiments of the present invention, cells of the
desired cell lineage can be isolated by using an affinity tag that
is specific for such cells. One example of an affinity tag specific
for a target cell is an antibody that is specific to a marker
polypeptide that is present on the cell surface of the target cell
but which is not substantially present on other cell types that
would be found in a cell culture produced by the methods described
herein.
[0114] The present invention also relates to kits, wherein the kit
comprises a basal salt nutrient solution and at least one compound
capable of stimulating ErbB2-directed tyrosine kinase activity. In
one embodiment, the kits comprise at least one ErbB3 ligand, as
described herein. In another embodiment, the kits comprise more
than one ErbB3 ligand. In another embodiment, the kits comprise at
least one of TGF-.beta. or a TGF-.beta. family member or a variant
or functional fragment thereof as described herein. In yet another
embodiment, the kits comprise more than one of TGF-.beta. or a
TGF-.beta. family member or a variant or functional fragment
thereof. In still another embodiment, the kits comprise at least
one fibroblast growth factor or variant or functional fragment
thereof. In another embodiment, the kits comprise more than one
fibroblast growth factor or variant or functional fragment thereof.
In a specific embodiment, the kits comprise at least one of FGF-7,
FGF-10, FGF-22 or variants or functional fragments thereof. In
another embodiment, the kits comprise serum albumin. In still
another embodiment, the kits comprise serum and/or at least one
insoluble substrate as described herein and/or at least one
disaggregation solution.
[0115] The kits of the invention may contain virtually any
combination of the components set out above or described elsewhere
herein. As one skilled in the art would recognize, the components
supplied with kits of the invention will vary with the intended use
for the kits. Thus, kits may be designed to perform various
functions set out in this application and the components of such
kits will vary accordingly.
[0116] Throughout this application, various publications are
referenced. The disclosures of all of these publications and those
references cited within those publications in their entireties are
hereby incorporated by reference into this application in their
entirety in order to more fully describe the state of the art to
which this invention pertains.
EXAMPLES
[0117] The human embryonic stem cell line BG01v (BresaGen, Inc.,
Athens, Ga.) was used in some of the experiments described herein.
The BG01v hESC line is a karyotypically variant cell line, which
exhibits stable karyotype containing specific trisomies (karyotype:
49, XXY,+12,+17). Parent cultures were maintained as described
previously (Schulz et al., 2003, BMC Neurosci., 4:27; Schulz et
al., 2004, Stem Cells, 22(7):1218-38; Rosler et al., 2004, Dev.
Dynamics, 229:259-274; Brimble et al., 2004 Stem Cells Dev.,
13:585-596). Briefly, the cells were grown in dishes coated with
MATRIGEL.TM. or fibronectin, in conditioned media from mouse
embryonic fibroblasts (MEFs) (MEF-CM) comprising DMEM:F 12 with 20%
KSR, 8 ng/ml FGF2, 2 mM L-Glutamine, 1.times. non-essential amino
acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM
.beta.-mercaptoethanol (Sigma, St. Louis, Mo., USA), with
collagenase passaging.
[0118] The defined culture (DC) media tested herein comprised
DMEM/F12, 2 mM Glutamax, 1.times. non-essential amino acids, 0.5
U/ml penicillin, 0.5 U/ml streptomycin, 10 .mu.g/ml transferrin
(all from Invitrogen, Carlsbad, Calif., USA) 0.1 mM
.beta.-mercaptoethanol (Sigma), 0.2% fatty acid-free Cohn's
fraction V BSA (Serologicals), 1.times. Trace Element mixes A, B
and C (Cellgro) and 50 .mu.g/ml Ascorbic Acid (Sigma). Variable
levels of recombinant growth factors were used, including FGF2
(Sigma), LongR3-IGF1 (JRH Biosciences), Heregulin-.beta. EGF domain
(HRG.beta., Peprotech), TGF.beta. (R&D systems), nodal (R&D
systems), LIF (R&D systems), EGF (R&D systems), TGF.alpha.
(R&D systems), HRG.alpha. (R&D systems), BMP4 (R&D
systems), and Activin A (R&D Systems). LongR3-IGF1 is a
modified version of IGF1 that has reduced affinity for IGF1 binding
proteins, some of which are expressed in hESCs. DC-HAIF is the
defined culture media as above, containing 10 ng/ml HRG-.beta., 10
ng/ml Activin A, 200 ng/ml LR-IGF1 and 8 ng/ml FGF2. DC-HAI is
defined culture media as above containing 10 ng/ml HRG-.beta., 10
ng/ml Activin A, and 200 ng/ml LR-IGF1. In both DC-HAIF and DC-HAI,
the LR-IGF1 component can, of course be replaced with IFG1.
[0119] MATRIGEL.TM. coated dishes were prepared by diluting Growth
Factor Reduced BD MATRIGEL.TM. matrix (BD Biosciences, Franklin
Lakes, N.J., USA) to a final concentration range of about 1:30 to
about 1:1000 in cold DMEM/F-12. In one embodiment, the
concentration of MATRIGEL.TM. is about 1:200. 1 ml/35 mm dish was
used to coat dishes for 1-2 hours at room temperature or at least
overnight at 4.degree. C. Plates were stored up to one week at
4.degree. C. MATRIGEL.TM. solution was removed immediately before
use.
[0120] For the tested conditions, parent cultures were plated into
6-well dishes for comparison of multiple conditions. Cultures were
typically plated directly into the test conditions. The cultures
were assessed every day and graded based on morphological criteria
4 to 5 days after plating. The grading scale of 1 to 5 involved
examining the whole culture and assessing overall proportion of
undifferentiated colonies, their relative size, and proportion of
colonies or parts of colonies exhibiting obvious differentiation.
Grade 5 indicates "ideal" cultures, with large undifferentiated
colonies and negligible differentiation. Grade 4 indicates a very
good culture, but with some obvious differentiation. Grade 3
indicates an acceptable culture, but with around half the colonies
exhibiting obvious differentiation. Grade 2 cultures are
predominantly differentiated, with occasional putative
undifferentiated cells. Grade 1 cultures contain differentiated
colonies or the cultures did not adhere or did not survive.
Cultures that exhibited good expansion of undifferentiated cells
were passaged to assess longer-term culture in these
conditions.
Example 1
Expression of ErbB1-3, Nrg1 and ADAM19 in BG01v Cells
[0121] Real time RT-PCR was used to demonstrate expression of
ErbB1-3, Neuregulin and ADAM-19 in BG01v cells (FIG. 1). BG01v
cells cultured in DC media as described above, containing 100 ng/ml
LongR3-IGF1 (LR-IGF1), 8 ng/ml FGF2 and 1 ng/ml Activin A were
harvested and RNA was prepared using the RNeasy mini kit (Qiagen)
according to the manufacturer's instructions. First strand cDNA was
prepared using the iScript kit (Biorad) and real time PCR was
carried out using a MJ Research Opticon thermal cycler.
[0122] TaqMan assays on demand (Applied Biosystems) for ADAM19
(HsO0224960_m1), EGFR (Hs00193306_m1), ErbB2 (Hs00170433_m1), ErbB3
(HsO0176538_m1), NRG1 (Hs00247620_m1), OCT4 (Hs00742896_s1) and
control GAPDH were used with TaqMan universal PCR (Applied
Biosystems). The real time amplification plots are shown in FIG. 1,
demonstrating expression of these transcripts in undifferentiated
BG01v cells.
Example 2
Inhibition of ErbB2 Slows Proliferation of BG10v Cells
[0123] The EGF domain family of ligands bind to the ErbB family of
receptor tyrosine kinases. To examine the effect of known
inhibitors of ErbB tyrosine kinases in hESCs, BG01v cells were
plated in 6 well trays on MATRIGEL.TM. diluted at 1:1000, in
defined culture medium (DC) containing 100 ng/ml LongR3-IGF1, 8
ng/ml FGF2 and 1 ng/ml Activin A. On the next day, DMSO (carrier
control), 50 nM-20 .mu.M AG1478 (an ErbB1 inhibitor), or 100 nM-20
.mu.M AG879 (an ErbB2 inhibitor) was added with fresh medium. The
cells were cultured for an additional 5 days, with daily media
changes. The cultures were then fixed and stained for alkaline
phosphatase activity.
[0124] Subconfluent colonies of AP+ BG01v cells observed (FIGS. 2A,
and B) in control and AG1478 cultured cells, indicating that
neither DMSO nor AG1478 (50 nM-20 .mu.M) had an apparent affect on
cell proliferation. AG879, however, substantially inhibited cell
growth at 5 .mu.M (FIG. 2C) and caused cell death at 20 .mu.M (not
shown). The cultures grown in AG879 did not appear to differentiate
and appeared to maintain a pluripotent morphology and alkaline
phosphatase activity, indicating that AG879 appeared to inhibit
proliferation without inducing differentiation, suggesting that
BG01v cells are reliant on ErbB2 signaling for cell survival.
Conversely, BG01v cells grown in similar conditions as above do not
appear to be reliant on ErbB1 signal for proliferation.
Example 3
BG01v cells are Maintained in Defined Media Containing
Heregulin
[0125] Expression of ErbB2 and ErbB3 and the inhibition of
proliferation with AG879 suggested that BG01v cells have active
endogenous ErbB signaling and that they may also respond to
exogenous HRG-.beta.. BG01v cells were grown in DC medium
containing 10 ng/ml HRG-.beta., 200 ng/ml LongR3-IGF1, 8 ng/ml FGF2
and 10 ng/ml Activin A, on MATRIGEL.TM. diluted 1:1000 (FIGS. 3A
and B). These cells were grown for 4 passages, or >20 days,
exhibited undifferentiated morphology and did not show elevated
spontaneous differentiation.
[0126] Furthermore, BG01v cells were also maintained for 2
passages, or >13 days, in DC medium comprising 10 ng/ml
HRG.beta., 200 ng/ml LongR3-IGF1, and 10 ng/ml Activin A. These
cultures proliferated normally and exhibited very low spontaneous
differentiation, demonstrating that BG01v cells could be maintained
in defined conditions with HRG.beta. in the absence of FGF2.
Example 4
The Role of ErbB2-Directed Tyrosine Kinase in ES Cells
[0127] RT-PCR demonstrated that mESCs express ADAM 19, Neuregulin1
(Nrg 1), and ErbB1-4 (FIG. 4A). In mESCs, ErbB2 and 3 appeared to
be expressed at higher levels than ErbB1, with low levels of ErbB4
being detected. These data suggest that endogenous HRG-.beta. could
be involved in driving mESC self-renewal.
[0128] The expression of the ErbB receptor transcripts in mouse
embryonic fibroblasts (MEFs) was also examined (FIG. 4B). MEFs are
a heterogenous population of cells derived from E12.5-13.5 viscera
that have been used historically to maintain mouse and human EC
cells and ES cells. Expression of Nrg1 and Adam19 in this
population suggests that the HRG-.beta. ectodomain is also present
in MEF-conditioned media and may exert significant effects upon
pluripotency.
[0129] AG1478 and AG879 were used to examine the role of HRG/ErbB
signaling in mouse ES cells. R1 mouse ES cells were maintained in
standard conditions in DMEM, 10% FBS, 10% KSR, 0.5 U/ml penicillin,
0.5 U/ml streptomycin, 1.times.NEAA, 1 mM sodium pyruvate, 1000
U/ml LIF (ESGRO), 0.1 mM .beta.-ME, and were passaged with 0.5%
trypsin/EDTA. 2.times.10.sup.5 cells/well were plated in 6 well
trays on MATRIGEL.TM. diluted at 1:1000. The day after plating,
DMSO (carrier control), 1-50 .mu.M AG1478, or 1-50 .mu.M AG879 was
added with fresh medium. The cells were cultured an additional 8
days, with daily media changes. The cultures were then fixed and
stained for alkaline phosphatase activity.
[0130] DMSO and 1-50 .mu.M AG1478 had no apparent affect on cell
proliferation, with subconfluent colonies of alkaline phosphatase
positive mESCs observed (FIGS. 5A-C). However, AG879 substantially
inhibited cell growth at 50 .mu.M (compare FIGS. 5D and 5F) and may
have slowed proliferation at 20 .mu.M (FIG. 5E). mESCs grown in
AG879 did not appear to differentiate and maintained a pluripotent
morphology, and alkaline phosphatase activity.
[0131] The results indicate that AG879 appeared to inhibit
proliferation, without inducing differentiation, of mESCs,
suggesting that mESCs require ErbB2 signaling for proliferation.
Conversely, mESCs do not appear to be reliant on an ErbB1 signal
for proliferation. The concentration of AG879 required to inhibit
proliferation was 10.times. higher for mESCs than that for BG01v
cells grown in defined conditions, indicating that either the serum
used in the mESC conditions may have interfered with the activity
of the drug, that AG879 has a lower affinity for the mouse ErbB2
tyrosine kinase than for human ErbB2 tyrosine kinase, or that ErbB2
may play slightly different roles with the different species of ES
cells.
[0132] Another highly selective inhibitor of the ErbB2 tyrosine
kinase, tyrphostin AG825 (Murillo, et al. 2001 Cancer Res 61,
7408-7412), was used to investigate the role of ErbB2 in human
ESCs. AG825 significantly inhibited proliferation of hESCs growing
in conditioned medium (CM) (FIG. 6A). AG825 inhibited proliferation
without widespread cell death, and viable hESCs could be maintained
for >5 days (not shown). Western blotting showed that AG825
inhibited autophosphorylation of ErbB2 at tyrosine-1248 in
starved/heregulin (HRG) pulsed hESCs growing in DC-HAIF (FIG. 6B).
Thus, disruption of ErbB2 signaling severely inhibited hESC
proliferation. To establish hESCs in defined growth conditions,
cultures could be passaged directly from CM conditions into DC-HAIF
and exhibited minimal spontaneous differentiation (FIG. 6C). Colony
and cell-counting assays confirmed that LongR3-IGF1 and HRG played
the major roles in self-renewal and proliferation in the context of
one of the embodiments of the present invention (FIG. 6D, 6E).
Phosphorylation of IGF1R, IR, FGF2.alpha., ErbB2, and ErbB3 was
also observed in both steady-state DC-HAIF cultures, and in starved
cultures that were pulsed with DC-HAIF (FIG. 6F).
Example 5
Culture of Mouse ES Cells in Defined Conditions
[0133] To further examine the role of HRG/ErbB2 signaling in mouse
ES cells, the proliferation of R1 ES cells was examined in DC
medium using a combination of growth factors. 1.times.10.sup.5
cells/well were plated in 6-well trays, coated with 0.2% gelatin,
in DC containing combinations of 10 ng/ml HRG-.beta., 100 ng/ml
LongR3-IGF1, 1 ng/ml Activin A, 1000 U/ml mouse LIF or 10 ng/ml
BMP4 (Table 1, below). Proliferation was observed over 8 days.
[0134] Viable colonies only grew in conditions containing at least
LIF/HRG-.beta. or LIF/BMP4 (Table 1). No additional obvious benefit
was observed when LongR3-IGF1 or Activin were added to these
combinations. Normal proliferation was observed in a control
parental culture, and no viable colonies were observed in defined
media without any growth factors.
TABLE-US-00001 TABLE 1 HRG IGF Activin LIF BMP4 Growth + No + + Yes
+ + No + + + Yes + + + No + + + + Yes + + No + + + Yes + + Yes + +
+ Yes
[0135] A quantitative assay was performed by plating
2.times.10.sup.5 cells/well in 6-well trays on 1:1000 MATRIGEL.TM.,
in selected combinations of 10 or 50 ng/ml HRG-.beta., 10 ng/ml
EGF, 1000 U/ml LIF or 10 ng/ml BMP4. The cultures were grown for 8
days, fixed, and the number of alkaline phosphatase colonies was
counted (FIG. 7A). No colonies were observed in defined conditions
without growth factors, and <45 colonies were observed with
HRG-.beta., HRG-.beta./EGF and HRG-.beta./BMP combinations. While
1358 colonies were observed in LIF alone, 4114 and 3734 colonies
were observed in the 10 ng/ml HRG-.beta./LIF and 50 ng/ml
HRG-.beta./LIF combinations, respectively. This indicated that in
defined conditions, LIF alone provided a substantial pluripotency
signal, and HRG-.beta. exhibited a large synergistic effect with
LIF, more than doubling the number of proliferating mESC colonies
in this assay. Low magnification images of this assay also indicate
this synergistic proliferative effect (FIGS. 7B-G).
Example 6
Characterization of Pluripotency of Human Embryonic Stem Cells
(hESCs) maintained in DC-HAIF
[0136] Multiple approaches were used to confirm the maintenance of
plasticity of hESCs in DC-HAIF. BG02 cells cultured in DC-HAIF for
6 months (25 passages) maintained the potential to form complex
teratomas (FIG. 8A) and representatives of the three germ layers in
vitro (FIG. 8B). Transcriptional analyses were used to compare
global expression in hESCs cells (Liu et al 2006, BMC Dev Biol 6,
20) maintained in CM and DC-HAIF. Greater than 11,600 transcripts
were detected in BG02 cells grown in DC-HAIF for 10 and 32
passages, and BG02 cells grown in CM for 64 passages. There were
about 10364 transcripts common to all populations (FIG. 8C),
including known hESC markers such as CD9, DNMT3, NANOG, OCT4, TERT
and UTF1 (not shown). High correlation coefficients were observed
in comparisons of CM and DC-HAIF cultures (R.sup.2select=0.928), as
well as in early and late passage cells (R.sup.2select=0.959) (FIG.
8D). Hierarchical clustering analysis demonstrated that BG02 cells
maintained in DC-HAIF grouped tightly and retained a close
similarity to BG02 and BG03 cells maintained in CM (FIG. 8E). These
data are consistent with previous analyses showing that
undifferentiated hESCs clustered tightly compared to embryoid
bodies or fibroblasts (Liu et al 2006, BMC Dev Biol 6, 20). Thus,
cells maintained in the compositions of the present invention are
able to maintain key markers of pluirpotency. Accordingly, the
compositions of the present invention can be used as a simple
medium for supporting self-renewal of differentiable cells.
Example 7
Maintenance of Human Embryonic Stem Cells (ESCs) on Humanized
Extracellular Matrices (ECMs) in DC-HAIF
[0137] To investigate the role of ErbB2 signaling and develop a
defined media for hESCs, DC-HAIF cultures were initially expanded
on culture dished coated with growth factor-reduced
MATRIGEL.TM.1:30, but could also be maintained successfully
long-term on this substrate diluted 1:200 (FIG. 9A), or 1:1000.
BG02 and CyT49 hESCs could also be maintained for >5 passages on
tissue culture dishes coated with human serum (FIG. 9B); human
fibronectin (FIG. 9C); or VITROGRO.TM. (FIG. 9D), which is a
proprietary humanized ECM.
Example 8
Single Cell Passaging of Human Embryonic Stem Cells (ESCs)
[0138] Multiple research groups have demonstrated that certain
triplodies, notably of hChr12 and 17, are accumulated in hESCs
under certain sub-optimal culture conditions (Baker et al., 2007
Nat. Biotech. 25(2):207-215). The appearance of triploidies seems
to be most directly related to poor cell survival when cultures are
split to single cells at passaging, providing a presumed strong
selective growth advantage for cells harboring these aneuploidies.
Conversely, hESCs growing in one embodiment of the present
invention, DC-HAIF, maintained high viability at plating after
being split to single cells (FIG. 10A-D). BG01 and BG02 cells
maintained a normal karyotype (FIG. 10E) after being passaged with
ACCUTASE.TM. for >18 and 19 passages respectively. The
maintenance of normal karyotype in cells demonstrated that
disaggregation of hESC cultures to single cells did not inherently
lead to the accumulation of these trisomies in hESCs maintained in
DC-HAIF. BG01 and BG02 cultures were also passaged by
disaggregation to single cells with multiple passaging agents (FIG.
11). Cultures were split with ACCUTASE.TM., 0.25% Trypsin/EDTA,
TrypLE, or VERSENE.TM. (EDTA) for 5 passages and karyotyped. The
data demonstrate that culturing and passaging hESCs in the
compositions of the present invention maintained a normal karyotype
in at least two human embryonic cell lines, using a variety of cell
disaggregation reagents.
[0139] Large-scale expansion of undifferentiated hESCs is also
possible, using the compositions of the present invention. A
starting confluent culture of BG02 cells in a 60 mm plate was
expanded in DC-HAIF through 4 passages to generate
>1.12.times.10.sup.10 cells in 20 days in a single experiment.
The cultures remained undifferentiated, as demonstrated by >85%
of the cells in the batch maintaining expression of markers of
pluirpotency such as OCT4, CD9, SSEA-4, TRA-1-81 when examined by
flow cytometry (FIG. 12A). Expression of other markers of
pluripotency was also observed by RT-PCR analysis, while markers of
differentiated lineages .alpha.-fetoprotein, MSX1 and HAND1 were
not detected (FIG. 12B). Fluorescence in situ hybridization
analysis demonstrated that the cells cultured and passaged in
DC-HAIF maintained expected copy numbers for hChr12 (98% 2-copy),
hChr17 (98% 2-copy), hChrX (95% 1-copy) and hChrY (98% 1-copy)
(FIG. 12C). Karyotyping analysis also demonstrated that a normal
euploid chromosome content and banding profile was maintained in
these cells
Example 9
Insulin and IGF1 Exert Different Effects on hESCs When Applied at
Physiological Concentrations
[0140] Essentially all of the reported culture conditions for hESCs
to date include supraphysiological levels of insulin, which can
stimulate both IR and IGF1R. To distinguish the activities that
insulin and insulin-substitutes exert, compared to IGF1, hESCs are
cultured in defined media conditions in physiological levels of
these growth factors. The concentrations of insulin and IGF1 are
titrated from about 0.2 to about 200 ng/ml and cell proliferation
is monitored by counting cells after 5 days. Cultures that expand
successfully are serially passaged 5 times. Physiological levels of
IGF1 support the expansion of hESC cultures, whereas physiological
levels of insulin do not, indicating that the activity of insulin
or insulin-substitutes cannot replace IGF1, and that IGF1 and
insulin (or insulin substitutes) represent separate classes of
biological activities with regard to action on hESCs.
Example 10
Methods for Screening the Effects of Supplements
[0141] To initially examine the effects of Vitamin B.sub.12 and
Vitamin B.sub.6 on the growth or differentiation hESCs growing at
an intermediate density, BG02 cells are split using ACCUTASE.TM.
and 1.times.10.sup.5 cells/well are plated in 6-well trays in
defined culture (DC) media. The DC media contains 10 ng/ml
HRG-.beta., 200 ng/ml LongR3-IGF1, and 10 ng/ml FGF10. Vitamin
B.sub.6 (0.5 .mu.M) and/or Vitamin B.sub.12 (0.5 .mu.M) are added
to experimental wells. Cell numbers in each condition are counted
after 7 days. Cell counting and colony counting of both
experimental and control wells will provide insight on the effects
of Vitamin B.sub.6 and Vitamin B.sub.12 on cell growth.
[0142] In addition, markers of differentiation, such as OCT4 can be
assayed in the experimental well to determine the effects of the
additives and supplements to the differentiation state of the
differentiable cells.
Example 11
Culturing of hESCs in the Absence of FGF2
[0143] BG02 cells were maintained long term in DC-HAI, for 20
passages (FIG. 13A), and BG01 cells were also serially passaged in
DC-HAI, both in the absence of FGF2. The cultures did not
deteriorate or exhibit overt differentiation, and exhibited normal
expansion of undifferentiated colonies throughout the culture
period. The maintenance of a normal male karyotype in a BG02
culture was demonstrated after 6 passages in DC-HAI (FIG. 13B,
20/20 normal metaphase spreads).
[0144] Transcriptional analyses were used to compare global
expression in hESCs cells maintained in DC-HAIF and DC-HAI. Total
cellular RNA was isolated from hESCs using Trizol (Invitrogen) and
was treated with DNase I (Invitrogen) according to the
manufacturer's suggested protocol. Sample amplification was
performed with 100 ng of total RNA using the Illumina RNA
Amplification kit and labeling was achieved by incorporation of
biotin-16-UTP (Perkin Elmer Life and Analytical Sciences) at a
ratio of 1:1 with unlabeled UTP. Labeled, amplified material (700
ng per array) was hybridized to Illumina Sentrix Human-6 Expression
Beadchips containing 47,296 transcript probes according to the
manufacturer's instructions (Illumina, Inc.). Arrays were scanned
with an Illumina Bead Array Reader confocal scanner and primary
data processing, background subtraction, and data analysis were
performed using Illumina BeadStudio software according to the
manufacturer's instructions. A minimum detection confidence score
of 0.99 (a computed cutoff indicating the target sequence signal
was distinguishable from the negative controls) was used to
discriminate the presence or absence of transcript expression. Data
analysis was performed using parallel approaches described for
other hESC samples (Liu et al 2006, BMC Dev Biol 6:20).
Hierarchical clustering was performed as described previously (Liu
et al 2005, BMC Dev Biol 6:20), and was based on average linkage
and Euclidean distances as the similarity metric using
differentially expressed genes identified by ANOVA (p<0.05).
Detailed descriptions of the sensitivity and quality control tests
used in array manufacture and algorithms used in the Bead studio
software are available from Illumina, Inc (San Diego, Calif.). The
majority of transcripts detected were expressed in both DC-HAIF and
DC-HAI BG02 cultures, including known hESC markers such as CD9,
DNMT3, NANOG, OCT4, TERT and UTF1 (not shown). High correlation
coefficients were observed in comparisons of DC-HAIF and DC-HAI
cultures (R.sup.2 select=0.961) (FIG. 14). Hierarchical clustering
analysis demonstrated that BG02 cells maintained in DC-HAI grouped
tightly and retained a close similarity to cells maintained in
DC-HAIF, as well as BG02 and other hESC lines in multiple culture
formats (FIG. 15). These data are consistent with previous analyses
showing that undifferentiated hESCs clustered tightly compared to
embryoid bodies or fibroblasts (Liu et al 2006, BMC Dev Biol
6:20).
[0145] Furthermore, BG02 cells maintained in DC-HAI differentiated
to representatives of mesoderm, endoderm and ectoderm in complex
teratomas formed in SCID-beige mice (not shown), formally
demonstrating the maintenance of pluripotency in cultures grown in
the absence of exogenous FGF2.
[0146] To examine if exogenous FGF2 was required in the context of
single cell passaging, BG01 cells were passaged with ACCUTASE.TM.
and grown in defined conditions containing only 10 ng/ml HRG-.beta.
and 200 ng/ml LongR3-IGF1 (DC-HI). These DC-HI cultures were
maintained for 10 passages, and did not exhibit overt
differentiation or a slowing of proliferation.
[0147] These studies clearly demonstrated that the provision of
exogenous FGF2 is not required when hESCs are maintained in defined
media minimally containing heregulin and IGF1. Furthermore cultures
absent FGF2 retained key properties of pluripotency, including
transcriptional profile and differentiation to mesoderm, endoderm
and ectoderm in vivo.
* * * * *